FAQs

The apoptosis, necrosis and cell viability assays are designed to stain dissociated cells in culture and have not been validated for organ culture. Annexin V staining of early chicken and mammalian embryos in culture has been reported in the scientific literature. For staining of living tissues, the specimen would need to be thin enough to allow exposure of the cells to the 36 kDa Annexin V protein. Also, damage to cell membranes from dissection or sectioning of tissues could result in high background staining.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

It has been reported in publications that concentrations of serum above 10% in the assay may affect the results.

See the following publications for more information

https://doi.org/10.2144/04373ST05

https://dx.doi.org/10.1007%2Fs10616-007-9057-4

No. Fixation, freezing, sectioning, or dissociation of tissues can affect the PS on the outer leaflet and compromise membrane integrity. Both Annexin V and Ethidium Homodimer III rely upon the presence of intact membranes in healthy cells to accurately distinguish healthy cells from apoptotic or necrotic cells. To detect apoptosis in fixed cells and tissues we recommend our TUNEL kits. Currently we are not aware of a fluorescent probe that specifically detects necrotic cells in fixed tissues. Necrotic cells in tissue sections are identified based on morphological criteria.

Yes, cells can be fixed with formaldehyde after staining. Because Annexin V staining is dependent on calcium, all buffers used for washing and fixation should contain 1.25 mM CaCl2. Fixation may increase the background signal from Ethidium Homodimer III. Wash the cells several times to remove unbound Ethidium Homodimer III before fixation.

Fixation is not recommended after staining with the Viability/Cytotoxicity Assay Kit (cat. # 30027) as the dead cell dye, EthD-III will transfer to all cells and not maintain dead cell-specific staining. Our Live-or-Dye™ NucFix™ Red (cat. # 32010) is a formaldehyde-fixable dead cell dye that can be used in bacteria.

Viability qPCR (vPCR) can be an alternative strategy for quantitating live and dead bacteria. Using dead cell-specific dyes, PMA (cat. # 40019) or PMAxx (cat. # 40069) that covalently modify DNA (of only dead cells) after photo-crosslinking, a simple quantitative PCR (qPCR) amplification is used to selectively amplify live-cell DNA. Learn more about the vPCR technology.

No, the concentration of Annexin V and Ethidium Homodimer III should be kept constant regardless of cell number. However, the staining concentrations can be increased or decreased if necessary to optimize staining.

The binding buffer is an isotonic buffer containing calcium, which is essential for the binding of Annexin V to phosphatidylserine.

The Annexin V protein that we use is a recombinant protein made in E. coli.

Annexin V is a 36 kDa protein that binds to the phospholipid phosphatidylserine. Therefore Annexin V binding is not species-specific.

The Live-or-Dye™ fixable viability stains, including Live-or-Dye NucFix™ Red, are dead cell stains that are amine-reactive. This property of the dyes makes staining compatible with fixation, but challenging in multi-cell layer systems such as 3D, Matrigel® or organoid cultures. As the dyes are reactive, the majority of the staining would be restricted to the outer/ exposed cell layer/s. Longer incubation times to allow the dye to penetrate deeper in to the cell layers would generally not be helpful as the labeling reaction is short-lived (15-30 min).

See our recommendations for dyes suitable for 3D cultures or organoids.

For fixable nuclear dead cell stains, other options to consider would be NucSpot® Far-Red. Designed to be an improved alternative to the more common dead cell stain 7-AAD, the dye is fixable to a degree. Although generally more suitable for flow cytometry as its spectral properties are not appropriate for standard fluorescence microscopy, imaging using a confocal may be better as the Ex/Em can be more appropriately matched. We would also recommend imaging soon after fixation and not storing the sample for extended periods post-fixation as staining would deteriorate over time.

The viability dye PMA may be another possibility. PMA, a photoreactive version of propidium iodide (PI), is generally used for viability PCR of bacteria. However, it can be used with mammalian cells (based on internal customer feedback) as fixable dead cell stain for flow cytometry. PMA is not fluorescent until it binds DNA. Subsequent exposure to bright light activates the dye and causes it to photo-crosslink to DNA, making it fixable. Incubation of the dye with cells would need to be done in the dark, and the excess dye washed away before exposing to light for photo-crosslinking. Optimization for light exposure sources (LED lights can be use to avoid heat) and light intensity (bright enough to crosslink the dye without photobleaching; generally close proximity to a bright LED or halogen lamp is required, room light is not sufficient) may be required.

Live-or-Dye NucFix™ Red is a reactive dye and most cell culture media, even if they are serum-free, contain a high concentration of reactive groups that would react with the dye, rendering it unreactive. While the dye would still get inside the cell and staining would be specific to the nucleus (the dye is fluorogenic and should only fluoresce when bound to DNA), it would not remain fixable.

PMA and PMAxx™ are photoreactive, cell impermeant DNA binding dyes that may be a better choice for fixable dyes for cell staining in medium. The dyes need blue light for photoactivation. Standard blue LEDs can be used for photocrosslinking of the dyes to DNA. If long-lasting fixation is not a requirement, and if the cells will not be undergoing extensive permeabilization, more fixable dead cell stains like NucSpot® Far-Red or 7-AAD  may be suitable options. Though the signal to noise is not as good as in unfixed cells, these dyes are more fixable than other dead cell dyes such as propidium iodide (PI).

Biotium’s TUNEL Assay Kits contain contain TdT (calf thymus), recombinant protein produced in E. coli.

You may wish to process floating and detached cells separately – collect the washes and spin them down and then stain those cells using the protocol for suspension cells. Alternatively you may collect floating cells, detach the adherent cells with trypsin (without EDTA), pool the cells together, and use the suspension cell protocol for staining.

Check to make sure your cell densities are in the linear range of the cell viability assay. Too few cells may fall below the limit of detection, and too many cells may saturate the OD. Perform an initial experiment to check the best linear range for your cell type and experimental set-up. You may need to vary the cell density assay incubation time. The kit protocols provide general guidelines and may need to be optimized for your experimental system.

The ViaFluor® SE dyes are susceptible to hydrolysis. Ideally the DMSO stock solution should be prepared on the day of use, with the intent that each vial supplied is for single use. If smaller aliquots are desired, it would be best to aliquot the initial DMSO stock, at the time of reconstitution, into single use quantities and store at -20°C, protected from light and moisture (desiccated), for up to a month. However, activity may be reduced over time. The dyes should only be added to aqueous buffer immediately before staining. The aqueous solution cannot be stored for reuse.

Our ViaFluor® SE Cell Proliferation assay is a dye dilution assay for cell division, like CFSE and CellTrace™ Violet from Thermo. This type of assay is commonly used to measure lymphocyte proliferative responses in culture and in vivo (if the labeled cells are injected back into mice). It requires flow cytometry to analyze and allows you to count how many cell divisions have occurred in the labeled cells.

For more information and a typical procedure for using fluorescent ViaFluor® SE Dyes with PMBCs, which can easily be adapted for use with other cell types, please see our Tech Tip: Measuring Cell Division in PMBCs by Flow Cytometry

If flow cytometry is not an option, we offer absorbance-based and fluorescence-based microplate assays for quantitating cell numbers. These measure mitochondrial activity (resazurin/MTT/XTT) or intracellular esterase activity (calcein AM) as a readout of viable cell numbers. Please visit the Cell Viability and Apoptosis technology page for more information.

The ATP-Glo™ assay is a luminescence assay for cellular ATP levels, which are proportional to the number of live cells. This assay requires a luminometer.

CellTrace is a trademark of Thermo Fisher Scientific.

Our Resazurin Cell Viability Assay (Cat. No. 30025) has red fluorescence (Ex/Em 530-560/590 nm), and is specifically designed for microplate reader. It is an economical, easy-to-use, and homogeneous (no-wash) assay for quantifying live cells. It is similar to alamarBlue®, PrestoBlue®, and CellTiter-Blue®.

The Calcein AM Cell Viability Assay (Cat. No. 30026) has green fluorescence (Ex/Em 485/530 nm), and also works well for microplate reader. This assay requires culture medium to be removed from cells before adding the viability dye in buffer. We also offer the Viability/Cytotoxicity Assay for Animal Live & Dead Cells, which combines calcein-AM with the fluorescent dead cell stain EthD-III, and is compatible with microplate reader.

MTT and XTT are colorimetric based assays, while resazurin can be measured using colorimetric or fluorescence detection. MTT is not a soluble product, so the cells must be lysed to solubilize the formazan salt before absorbance can be measured. XTT and resazurin do not require cell lysis, allowing kinetic monitoring of the same samples at different timepoints.

AlamarBlue® contains resazurin and additional compounds to prevent the over-reduction of resazurin to a non-fluorescent product. These additives also slow the rate of generation of the fluorescent product. Consequently, the alamarBlue® assay requires longer incubation times compared to resazurin.

Resazurin is reduced by cells to the fluorescent product resorufin. Resorufin can be reduced further to a non-fluorescent compound. Therefore the densest wells may have lowest fluorescence due to over-reduction of the substrate. Please see the product information sheet for more details. The kit protocol provides general guidelines and may need to be optimized empirically for your experimental system. You may need to vary cell density or assay incubation time to ensure that your samples fall in the linear range of the kit.

Medium with phenol red is compatible with the resazurin assay. Phenol red does not interfere with the resazurin reaction nor does it affect detection.

CellBrite® Cytoplasmic Membrane Stains are lipophilic dyes for simple, non-toxic, stable labeling of membranes in live or fixed cells. Cells can be fixed with formaldehyde before or after CellBrite® staining. But the staining has poor tolerance for permeabilization or methanol fixation, so CellBrite® staining is not easily combined with intracellular immunofluorescence (IF) staining. The dyes also do not stain bacteria or yeast. CellBrite® NIR dyes are CellBrite® dyes with near-infrared fluorescence compatible with small animal NIR imaging systems.

CellBrite® Fix and MemBrite® Fix stains were developed to overcome some of these shortcomings. They are novel covalent stains that can be fixed and permeabilized for IF staining. CellBrite® Fix Membrane Stains are fluorogenic reactive membrane dyes that rapidly accumulate at the plasma membrane. When they incorporate into lipids, they become fluorescent, and at the same time react covalently with membrane proteins for stable labeling. Staining takes only 15 minutes in a single step with no wash. CellBrite® Fix stains mammalian cells, yeast, and bacteria.

MemBrite® Fix Cell Surface Stains do not bind lipids, but label cell surface proteins. MemBrite® Fix requires a two-step staining protocol with washing, but offers a more extensive choice of dye colors than CellBrite® Fix. MemBrite® Fix also can be used to stain yeast. But unlike original CellBrite® dyes and lectins, CellBrite® Fix and MemBrite® Fix cannot be used on cells that are already fixed.

To select a dye that’s right for your application, see our Membrane and Cell Surface Stains Comparison, or download our Membrane & Surface Stains Brochure.

CellBrite® Cytoplasmic Membrane Dyes do not efficiently stain EVs. Some of the CellBrite® Fix, MemBrite® Fix, and CellBrite® Steady dye options can be used for this application. However, for optimal staining of exosome membranes we recommend our ExoBrite™ True EV Membrane Stains, which are novel lipophilic membrane dyes specifically designed and optimized for efficient staining of EV membranes with minimal dye aggregation. See our Extracellular Vesicle Research page for more information about our complete line of EV stains and antibodies

Biotium’s CellBrite® Cytoplasmic Membrane Dyes are dye delivery solutions that can be added directly to normal culture media to uniformly label suspended or adherent cells in culture. The PKH dyes are structurally related dyes for cell membrane labeling. But unlike CellBrite®, labeling with PKH dyes requires multiple steps and subjects cells to an iso-osmotic mannitol loading medium that can negatively affect cell membrane integrity and viability.

CellBrite® Fix dyes also feature rapid and simple labeling in isotonic buffer. Cells can be fixed and permeabilized after labeling with CellBrite® Fix, unlike original CellBrite® dyes or PKH dyes, which don’t tolerate detergent.

DiI (DiIC18(3); 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine) is a widely used lipophilic carbocyanine dye that labels cell membranes by inserting its two long (C18) hydrocarbon chains into the lipid bilayer. It has been extensively used for the antero- and retro-grade labeling of neurons.

Neuro-DiI (also available as a ready-to-use staining solution, CellBrite® Green) was developed by Biotium as an alternative to DiI. It has two additional t-butyl groups (one on each side of the dye) attached to the dye chromophore which introduce structural flexibility in the dye making it much more soluble in both organic solvents and in cell membranes. Improved dye solubility in the membranes enables rapid dispersion of the dye molecules, away from each other much more rapidly than regular DiI which has a relatively flat structure that promotes dye aggregation, and slows down dispersion of dye molecules. Neuro-DiI therefore results in more uniform, rapid and stable staining as compared to DiI.

FAST™ DiI uses a different strategy to solubilize the dye. Replacing the saturated C18 lipophilic chain of regular DiI with unsaturated linoleyl chains gives the dye molecules a bent structure, making them less likely to aggregate and thus disperse faster in the membrane.

Both Neuro-DiI and FAST™ DiI would diffuse faster than regular DiI in membranes, but a comparison of the relative diffusion rate between the two dyes has not been made. Neuro-DiI may have a potential advantage over FAST™ DiI. The t-butyl groups of Neuro-DiI render it more hydrophobic than FAST™ DiI, likely resulting in more stable membrane staining and make dye transfer between cells less likely.

Original CellBrite® Cytoplasmic Membrane Stains are lipophilic carbocyanine dyes. These dyes undergo an increase in fluorescence when they insert into lipid bilayers. Lipophilic carbocyanine dyes stably label the plasma membrane and other intracellular membranes of cells. They also can be used to stain fixed cells or artificial lipid bilayers. CellBrite® cytoplasmic membrane stains are lipophilic carbocyanine dyes. These dyes undergo an increase in fluorescence when they insert into lipid bilayers. Lipophilic carbocyanine dyes stably label the plasma membrane and other intracellular membranes of cells. They also can be used to stain artificial lipid bilayers. Immediately after staining cultured cells, the dyes primarily localize to the plasma membrane. If cells are cultured over time after staining, the labeled membranes are internalized and staining gradually becomes mostly intracellular.

Lipophilic carbocyanine dyes like our original CellBrite® Cytoplasmic Membrane Stains have been used to stain neuronal cells in culture for several weeks, and in vivo for up to a year (see note below). The dyes do not appreciably affect cell viability, and do not readily transfer between cells with intact membranes, allowing cell migration and tracking studies in mixed populations. Staining with the covalent stains CellBrite® Fix and MemBrite® Fix lasts up to 48 hours in tissue culture cells (see note below).

Note: Over time, all cell surface stains will be internalized and become intracellular as membranes turn over by endocytosis. The rate of internalization may vary by cell type, rate of membrane turnover, and rate of cell division. In immortalized cells in culture, most of the surface staining becomes internalized over the course of about 24 hours for CellBrite®, CellBrite® Fix, and MemBrite® Fix stains.

For an alternative stable and fixable stain for long term cell tracking or tracking cells in mixed cultures, see our ViaFluor® SE Cell Proliferation Dyes. These dyes covalently label intracellular proteins throughout the cell and are non-toxic and fixable.

Cells can be fixed with formaldehyde after labeling with the original CellBrite® or CellBrite® NIR cytoplasmic membrane dyes. Permeabilization of cells with detergents or solvents, or mounting medium containing glycerol may adversely affect staining. Permeabilization with digitonin (10 ug/mL to 1 mg/mL) has been reported to be compatible with lipophilic carbocyanine dye staining. We’ve seen good results when formaldehyde-fixed cells are permeabilized before staining with CellBrite® dyes (See Tech Tip: Combining Lipophilic Membrane Dyes with Immunofluorescence).

The CF® Dye WGA Conjugates can also be used to label the cell surface or plasma membrane in fixed cells/tissues or FFPE sections. On tissue sections however, the lectins would label glycoproteins on all cell membranes, external (plasma membrane) as well as internal (organelle membranes). WGA staining can also be tissue- and cell-type dependent i.e. it is based on the expression pattern of glycoproteins on the cell membranes.

CellBrite® Fix Membrane Stains and MemBrite® Fix Cell Surface Stains belong to a new class of membrane dyes designed to covalently label the cell surface. They can withstand fixation and permeabilization, or fixation with alcohol after labeling of live cells. CellBrite® Fix and MemBrite® Fix cannot be used to label the plasma membranes of fixed cells or tissues (the dyes label the cytoplasm in fixed cells).

To find the right dye for your workflow, see our Comparison of Membrane & Cell Surface Stains, or download our Membrane & Surface Stains Brochure.

Lipophilic carbocyanine dyes like CellBrite® and CellBrite® NIR Cytoplasmic Membrane Dyes can be used to stain formaldehyde-fixed cells. Fixation with methanol or other solvents is not recommended. Permeabilization with detergents after staining will extract the dyes and alter the staining pattern. However, we’ve seen good results when formaldehyde-fixed cells are permeabilized before staining with CellBrite® dyes (See Tech Tip: Combining Lipophilic Membrane Dyes with Immunofluorescence).

CellBrite® Fix, MemBrite® Fix, and CellBrite® Steady cannot be used to stain the plasma membrane of fixed samples; these dyes will primarily stain intracellular structures in cells that are already fixed.

To find the right dye for your workflow, see our Comparison of Membrane & Cell Surface Stains, or download our Membrane & Surface Stains Brochure.

The loading buffer in the CellBrite® Blue kit is required for solubilizing the DiB dye solution. The other CellBrite® dyes are more soluble and do not require this.

It’s common for DiB Loading Buffer (30024B) to solidify into a gel during storage. This does not affect the product, but the buffer must be in liquid form before use. Heat the solidified gel to 50-60°C for 5-10 minutes and vortex periodically until it has formed a clear liquid. DiB Loading Buffer is viscous, so pipet it slowly to ensure the correct volume is added.

You can heat the DiB Cell Labeling Solution to 37°C for 10 minutes or longer, and pipette the solution up and down or vortex to mix completely until is is completely dissolved.

CellBrite® dyes are ready-to-use solutions of the lipophilic carbocyanine dyes DiI, DiO etc. It is challenging to get uniform labeling with CellBrite® dyes due to their hydrophobicity, CellBrite® Red (#30023), which is DiD,  is particularly difficult as the hydrophobic dye tends to aggregate and give uneven staining. Warming the dye vial to ~50°C to dissolve any dye aggregates before staining could be tried. Also rather than adding the dye directly to the medium, we recommend diluting in it medium (1:200 dilution) first, and then adding the medium with the dye to the cells. Also, be aware that these dyes will be internalized if cells are cultured after staining.

Alternatively, for live cell staining, CellBrite® Steady or the reactive dyes CellBrite® Fix or MemBrite® Fix would be more suitable as they result in more even staining.

CellBrite® dyes are not compatible with glycerol-based mounting media as this results in altered staining and high background. Organic mounding media are also not suitable. We recommend imaging in directly in PBS (or other aqueous buffer). Coverslips should be mounted using PBS and sealed with a suitable coverslip sealant such as CoverGrip™ or nail polish. Stained samples can be stored in PBS at 4°C for several weeks or longer.

While CellBrite® Cytoplasmic Membrane Dyes can stain formaldehyde-fixed cells, they generally do not give good results in cryosections, possibly because the cell membrane integrity is disrupted, exposing other membrane structures to the dyes. Some customers have reported success using these dyes with vibratome sections.

CellBrite® Cytoplasmic Membrane Dyes are not suitable for membrane staining in FFPE samples as membrane lipids are extracted during the dewaxing and rehydration process. Similarly, acetone or methanol fixation of cryosections will extract lipids, leading to poor staining.

CellBrite® Fix, MemBrite® Fix, and CellBrite® Steady are recommended for use on live cells only. In fixed cells or sections they will label intracellular structures.

In some tissue types, lectins such as CF® Dye WGA Conjugates, CF® Dye Concanavalin A Conjugates, or CF® Dye PNA Conjugates may be useful for staining cell boundaries in FFPE or frozen sections. However, the staining pattern of lectins is highly dependent on cell and tissue type, so we recommend consulting the literature before trying these stains for your tissue of interest.

Alternatively, immunostaining using cell surface-specific antibodies could be done.

So far we have not found a universal plasma membrane stain for tissue sections. This is an application of interest to us and our customers, so we are working to find new solutions.

The lipophilic CellBrite® dyes would not be suitable for solvent-cleared samples as membrane lipids would be extracted during the solvent treatment step. The reactive membrane dyes, CellBrite® fix and MemBrite® too would not work as these are live cell stains, and in dead/ fixed samples, they would stain all accessible targets resulting in whole cell staining.

However, if staining of live cells is possible, the reactive CellBrite® fix and MemBrite® fix membrane dyes may be used before fixation and solvent-clearing, but these dyes may not penetrate multilayer cells or tissues effectively.

Staining using CF® Dye WGA Conjugates may be an option, but staining can be cell- and tissue-type dependent i.e. it is based on the expression pattern of glycoproteins on the cell membranes. You may verify is WGA staining is an option for your cell or tissue type based on published literature. It is also important to note that if tissue sections are used, the lectins would stain glycoproteins on all cell membranes, external (plasma membrane) as well as internal (organelle membranes).

Alternatively, immunostaining using cell surface-specific antibodies could be used.

To find the right dye for your workflow, check out the links below:

Tech Tip: Cell Surface Stains for Live & Fixed Cells

Comparison of Membrane & Cell Surface Stains

Or download our Membrane & Surface Stains Brochure.

Lipophilic carbocyanine dyes like CellBrite® Cytoplasmic Membrane Dyes may stain tissue slices or organotypic cultures, but due to their lipophilicity, they probably will not penetrate through multiple cell layers in a thick tissue slice. The dyes have been used for cell tracing in organoids and labeling hydrogel cultures. However, for this application, usually the cells are labeled before they are placed in organoid culture or embedded in hydrogel.

This class of dyes have been widely used for neuronal tracing in organisms, tissue slices, Drosophila embryos, and chicken embryos by microinjection or spot-labeling with crystals of solid dye. Neuronal tracing also can be done in formaldehyde-fixed tissues or embryos.

See our Tech Tip: Researching Applications for Membrane Dyes for more information and selected references.

Matrigel is a registered trademark of Corning Inc.

Lectins such as CF® Dye WGA Conjugates, CF® Dye Concanavalin A Conjugates, or CF® Dye PNA Conjugates can be used to label the cell surface or plasma membrane in FFPE or frozen sections. However lectins would label glycoproteins on all cell membranes, external (plasma membrane) as well as internal (organelle membranes). Lectin staining also is tissue- and cell-type dependent, based on the expression pattern of glycoproteins on the cell membranes. Therefore they generally do not selectively label all cell outlines or boundaries in a tissue section.

Other cell surface membrane stains such as the lipophilic CellBrite® or CellBrite® NIR dyes are not suitable for membrane staining in FFPE samples as membrane lipids are extracted during the dewaxing and rehydration process. Similarly, acetone or methanol fixation of cryosections will extract lipids, leading to poor staining.

CellBrite® Cytoplasmic Membrane Dyes may stain formaldehyde-fixed cryosections. However, because the cell membrane integrity is disrupted in frozen sections, CellBrite® dyes probably will not selectively stain the plasma membrane in tissue sections and will likely stain intracellular structures as well.

The reactive, fixable membrane stains, CellBrite® Fix and MemBrite® Fix are for live cell staining only. In fixed sections, they would indiscriminately react with target proteins throughout the cell, staining the entire cell.

Alternatively, immunostaining using cell surface-specific antibodies could be done.

To find the right dye for your workflow, see our Comparison of Membrane & Cell Surface Stains, or download our Membrane & Surface Stains Brochure.

CellBrite® and MemBrite® Stains were originally developed for staining mammalian cells in culture, but some of the stains also have been validated for other organisms and applications. For dyes to stain yeast or bacteria membranes, see Cellular Stains in Different Organisms. For information on staining other organisms or cell types, please see our Tech Tip: Researching Applications for Membrane Dyes.

The CellBrite® Cytoplasmic Membrane Dyes do not stain bacteria. The reactive CellBrite® Fix dyes stain both gram-positive and gram-negative bacteria, while the MemBrite® Fix dyes stain only gram-positive bacteria. However we have not tested these dyes for cell division tracking in bacteria.

There is literature describing the use of CFSE to track bacterial cell division,  the ViaFluor® SE cell proliferation dyes are likely to work in a similar manner, but we have not tested this.

See our Cellular Stains Table for a comprehensive list of cellular stains with their ability to stain various cell types.

Yes, CellBrite® Cytoplasmic Membrane Dye staining can be done in buffer instead of culture medium. If you are staining adherent cells, we recommend using a buffer like HBSS with calcium/magnesium, which helps maintain cell adherence and morphology.

CellBrite® and MemBrite® Stains were originally developed for staining mammalian cells in culture, but some of the stains also have been validated for other organisms and applications. If you are staining a non-mammalian cell type or organism that grows in water or other medium, please see our Tech Tip: Researching Applications for Membrane Dyes. For dyes to stain yeast or bacteria membranes, see Cellular Stains in Different Organisms.

Yes, CellBrite® Cytoplasmic Membrane Dye staining can be done at room temperature or lower, instead of 37°C, this may reduce dye internalization during staining. Be aware that staining at 4°C may result in dye precipitation or uneven staining.

CellBrite® and MemBrite® Stains were originally developed for staining mammalian cells in culture, but some of the stains also have been validated for other organisms and applications. If you are staining a non-mammalian cell type or organism that grows at a temperature below 37°C, please see our Tech Tip: Researching Applications for Membrane Dyes. For dyes to stain yeast or bacteria membranes, see Cellular Stains in Different Organisms.

See below for our recommended alternatives to Akoya tyramides. Unless noted as a direct replacement, these tyramide dyes are spectrally similar alternatives. They have not been validated as direct replacements for use with Akoya kits or imaging systems.

CF® Dye Alternatives for Akoya Tyramides

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF® Dye succinimidyl ester stability

Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Dyes that carry multiple negative charges can introduce background. Usually, this is more of a concern with labeled antibodies that carry many dyes, as opposed to a small toxin like bungarotoxin. When staining tissues, the endogenous autofluorescence of the tissue itself is often the most significant source of background. Endogenous fluorescence background in tissue is usually highest in the blue wavelengths (DAPI channel) and lowest in the far-red (Cy®5 channel). Our CF®633 bungarotoxin (catalog no. 00009) is a far-red conjugate for the Cy®5 channel with a low negative charge that should have low background from either the dye or autofluorescence.

We test fluorescent bungarotoxin on rat skeletal muscle sections. While the tissue shows autofluorescence, the bungarotoxin staining of motor endplates is usually much brighter than the background for all of the dye colors we’ve tested.  However, if you are staining human tissue (especially brain), lipofuscin autofluorescence may be bright in all channels. This usually shows up as bright, punctate dots around cell nuclei. While we would usually recommend our TrueBlack® lipofuscin quenchers for human brain tissue, they are not compatible with bungarotoxin staining. We have, however, found that EverBrite TrueBlack® Mounting Medium (cat. no. 23017) can be used to mount skeletal muscle sections stained with bungarotoxin.

Cy Dye is a registered trademark of Cytiva.

See our CF® Dye Quick Reference Table for a list of dyes and summary of their features. Our CF® Dye Selection Guide has more detailed information on each CF® dye, and ordering information for our various CF® dye product lines. You can download CF® dye normalized absorption and emission data in Excel format.

The exact chemical structures of CF® dyes are currently confidential but will be fully disclosed at a later stage when pending patents become granted. In general terms, the structure of a CF® dye may be divided into two parts: a) dye core structure (i.e. the aromatic ring skeleton that defines the dye’s color or absorption/emission wavelengths), and b) core structure-modifying elements. At present, CF® dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes. Blue fluorescent CF® dyes are based on coumarin or pyrene dye core structure, while green to near-IR CF® dyes are based on either cyanine or rhodamine dye core structures. Core structure-modifying elements refer to various chemical attachments to the core structure and are a key aspect of the CF® dye invention that makes CF® dyes superior to other commercial dyes.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Some products are shipped with blue ice packs as an extra precaution against high temperatures. The blue ice packs may be thawed upon arrival without affecting product performance.

Products with recommended storage at ultra low temperature (-70°C) that also ship on dry ice should arrive frozen. If a product you received was shipped on dry ice and thawed during transit, please contact customer service at order@biotium.com.

You can also download our Product Storage Statement here.

Most CF® dyes carry 1-2 negative charges while a few cyanine-based near-IR CF® dyes carry 3-4 negative charges. However, the more negatively charged CF® dyes comprise unique structural features that shield the negative charges such that the biomolecules (such as antibodies) the dyes label do not lose specificity due to the excessive negative charges.

CF® initially was an abbreviation for Cyanine-based Fluorescent dyes. These were the first patented CF® Dyes based on cyanine dye structures. Since then, our CF® Dye patent portfolio has expanded to include four different fluorescent dye core structures that cover the fluorescence spectrum from UV to NIR. Today, we believe “CF” more aptly stands for Clear Fluor: dyes that produce superior signal-to-noise.

CF® dyes are highly water soluble (>100 mg/mL). They are also very soluble in other polar solvents, such as DMSO, DMF, methanol and ethanol. However, CF® dyes are poorly soluble or insoluble in non-polar solvents.

Rhodamine-based CF® dyes (designated R) generally have better photostability but weaker fluorescence than their cyanine-based equivalents (designated C). Therefore, rhodamine-based near-IR CF® dyes are a better choice for microscopy, while cyanine-based CF® dyes are more ideal for flow cytometry, Western blotting, and other applications where photobleaching is less of a concern. Another factor to consider is the size of the dyes. Some of the cyanine-based near-IR CF® dyes are much larger than the rhodamine-based equivalents. For antibody labeling, either version of the CF® dyes is suitable. However, for applications where the dye size may cause a steric problem, the smaller dye may be a better choice.

We don’t have direct experience or customer feedback on CF dyes for 6-color imaging on LSM 800, but because this instrument has tunable excitation and linear unmixing, you should have a lot of flexibility in dye choice. We would also recommend contacting Zeiss support to see if they have advice for selecting dyes for multi-color experiments for the instrument.

The violet excited 405/430 dyes are generally less bright and should be used for more abundant targets. The 754 dye would also be recommended for more abundant targets. Even with optimal excitation, the GaAsp detectors on the Zeiss LSM 880 are expected to be less responsive to NIR wavelengths. The other dyes are very bright and would be better for less abundant targets to minimize cross-talk.

Below we’ve listed the dyes we’d usually recommend for the standard laser lines on the LSM 880.

405 nm laser line

CF®405S, Ex/Em 404/431 nm; recommended for abundant targets

458 nm laser line

CF®430, Ex/Em 426/498 nm; recommended for abundant targets

488 nm laser line

CF®488A, Ex/Em 490/515 nm; bright and photostable

Note: CF®488A and CF®514 must be separated by spectral imaging if used together

514 nm laser line       

CF®514, Ex/Em 516/548 nm; dye for spectral imaging

Note: CF®488A and CF®514 must be separated by spectral imaging if used together

561 nm laser line

CF®568 Ex/Em 562/583 nm; very bright and photostable

Note: CF®568 and CF®594 must be separated by spectral imaging if used together

594 nm laser line

CF®594, Ex/Em: 593/614 nm; very bright and photostable

Note: CF®568 and CF®594 must be separated by spectral imaging if used together

633 nm laser line

CF®640R, Ex/Em 642/662 nm; CF®640 will be brighter with on-center excitation

CF®660R, Ex/Em 663/682 nm; CF®660R is off-center for this laser, but very bright and extremely photostable; less likely to have cross-talk with CF®594

Tunable 2P Laser 720-1020

CF®754, Ex/Em 745/786; recommended for abundant targets (LSM800 detectors are expected to be less sensitive for NIR dyes)

All three of these dyes can be excited by the 405 nm laser (or UV mercury lamp). They differ in their emission wavelengths. CF®405S has blue fluorescence emission at 431 nm, similar to AlexaFluor® 405, Cascade Blue®, and DyLight™ 405 (Thermo Scientific). CF®405M has longer wavelength blue fluorescence emission at 452 nm, similar to Horizon™ V450 (BD Biosciences) and Pacific Blue® (Thermo Scientific). CF®405L has orange fluorescence emission at 545 nm, similar to Pacific Orange® (Thermo Scientific). We recommend choosing the dye that best fits your instrument’s detection settings. See our CF® Dye Quick Reference Table and CF® Dye Selection Guide for more information on CF® dyes.

There are usually three aspects to dye stability: 1) chemical stability of the dye core structure; 2) stability of the reactive group; and 3) photostability of the dye.

Chemical stability of the dye core structure:

This refers to resistance of the dye core structure to decomposition caused by factors other than photo-bleaching. These factors may include temperature, pH and incompatibility with other chemicals in the medium. This type of stability information is most useful for estimating the shelf-life of the dye that is already covalently attached to another molecule (e.g., an antibody), or for assessing the chemical compatibility of the dye in certain applications. CF® Dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes, all of which are known to have excellent chemical stability. In general, CF® Dyes are far more stable than the antibodies they label. Thus, if a CF®-labeled antibody loses activity during storage, the problem is not likely to be caused by the dye. CF® Dyes are also stable enough for labeled nucleic acids to be used in PCR or nucleic acid hybridization, where high temperature is involved.

Stability of the reactive group:

Reactive CF® Dyes comprise a reactive group used in bioconjugation. Among the various reactive groups, only amine-reactive succinimidyl ester (SE) and thiol-reactive maleimide groups are unstable because the small amount of moisture trapped in or leaked into the packaging vials can cause hydrolysis of the reactive groups over time. The SE group, in particular, is susceptible to degradation. Thus, in order to slow degradation, CF® Dyes comprising these reactive groups must be stored at -20°C under anhydrous conditions. Furthermore, stock solutions of the dyes must be made using dry solvents, such as anhydrous DMSO. One advantage of CF® Dye SE products over other commercial dyes is their relatively high stability. Normally, an SE group can be derived from either an aliphatic or an aromatic carboxylic acid group, but an aliphatic carboxylic group tends to result in a more stable SE, offering higher resistance to hydrolysis and thus better labeling efficiency. All of the CF® SE Dyes have their SE groups derived from aliphatic carboxylic acid groups, unlike many commercially available SE dyes that are often prepared from aromatic carboxylic acid groups.

Photostability:

This refers to the dye’s ability to withstand photobleaching. For most dyes, photostability is not a major problem for routine handling under ambient light or for applications, such as flow cytometry and Western blotting, where the dyes are only briefly exposed to light. However, for microscopy, especially for confocal microscopy, where the dyes may be subject to intense illumination for an extended period of time, photobleaching can be a major concern. Similar to the photostability of other fluorescent dyes, both the dye core structure and the structure-modifying groups attached to it play a role in the photostability of CF® Dyes. CF® Dyes bear the core structure of rhodamine, cyanine, pyrene or coumarin dyes; among the four types of core structures, rhodamine core is the most photostable, followed by cyanine and then by pyrene and coumarin cores. The structure-modifying groups and the way they are attached to the dye cores are a key innovative aspect of CF® Dye technologies that contributes to the superior photostability of CF® Dyes over that of other commercial dyes. In general, rhodamine-based CF® Dyes, whose wavelengths range from visible to the near-IR region, offer the best photostability, making the dye ideal for microscopy applications.

CF® dyes are chemically stable within the pH range of at least 2 –11. The fluorescence of most CF® dyes is relatively insensitive to pH, except for that of CF®405M, CF®568, CF®620R, and CF®633. The fluorescence of these four CF® dyes becomes weaker when pH drops below 4.5.

CF® dyes can tolerate formaldehyde fixation. However, whether a CF® dye-labeled probe is fixable will depend on the fixability of the probe itself. Proteins with free amine groups that bind other proteins generally are formaldehyde-fixable.

There is no simple answer to this question as the quantum yield of a fluorescent dye can vary widely, depending on the dye’s micro-environment. For example, the quantum yield of a dye attached to a protein may be very different from the quantum yield of the free dye. For dyes attached to a protein, the quantum yield is highly dependent on how many molecules of the dye are attached to the protein (i.e. degree of protein labeling). In general, a higher degree of protein labeling leads to a lower dye quantum yield due to fluorescence quenching via dye-to-dye interaction. For this reason, as the degree of labeling increases, fluorescence intensity of the labeled protein will eventually reach a maximum and start to decline thereafter. In fact, one of the best ways to compare the relative quantum yields of different dyes is to plot the total fluorescence of the labeled proteins as a function of degree of labeling by the dyes as we have done with CF® dyes and other commercial dyes. CF® dyes generally give higher slopes than other commercial dyes in the plots, suggesting less quantum yield decline with increasing degree of protein labeling.

Lifetime data for several CF® dyes are available, both for the free dye in buffer as well as for the corresponding CF® dye conjugated secondary antibody. These are listed below.

Measurements were made on a Stellaris 8 STED FALCON microscope courtesy Leica Microsystems, Germany. § Fluorescence lifetime measurements of CF® dye labeled anti-mouse secondary antibodies used for immunostaining microtubules in U2OS using mouse anti-tubulin (DM1a) and mounted in ProLong™ Diamond. *Lifetime data obtained via customer communication under different experimental conditions and imaging setup.

CF® dyes are ideal for protein labeling because of their high water solubility, which reduces fluorescence quenching. They are also useful for labeling oligonucleotides that require multiple copies of a dye for maximal fluorescence, such as the preparation of FISH probes, where water soluble dyes can minimize fluorescence quenching. Finally, CF® dyes make excellent polar tracers that can be used for visualizing the morphology or long-term tracing of neurons after microinjection. CF® dyes and their conjugates are ideal for fluorescence microscopy applications, flow cytometry, Western blotting, and in vivo imaging (near-IR CF® dyes).

Reactive dyes for bioorthoganol conjugation (click chemistry) including alkyne, azide, BCN, TCO, and methyltetrazine can be used to prepare polymers. We do not offer CF dye methacrylate at this time, please contact techsupport@biotium.com to inquire about custom synthesis.

Yes, CF® dye tyramides can be used for in situ hybridization using Tyramide Signal Amplification¹ (TSA). They are also compatible with the RNAscope assays  for RISH².

¹ doi: https://doi.org/10.1038/s41598-018-38171-5.

² doi: http://dx.doi.org/10.1101/651083.

If you order an oligo to be synthesized with an amino group on one end, then our CF® Dye succinimidyl ester (SE) dyes could be used to label it using a standard amine labeling protocol for DNA.

We recommend using an HPLC purified oligo, with an amino group with a C6 linker at the 5′ or 3′ end. To remove free dye after labeling, you could use G25 Sephadex, gel purification, ethanol precipitation, or HPLC, depending on the degree of purity needed.

Alternatively, this may be something that an oligo synthesis company may be able to perform for you as a custom labeling.

We do not have firsthand experience with LPS labeling, but according to the literature,  LPS has been labeled using amine-reactive dyes, like FITC. Our amine-reactive CF® Dye Succinimidyl Esters should also work for this. There is a publication for enzymatic labeling of LPS using dye hydrazides. Our CF® Dye hydrazides could be used in this method. The paper also describes the traditional amine labeling method and purification of the conjugate.

For dyes or reagents that are supplied lyophilized (as solids), it is hard to compare quantities based on appearance of the dye in the tube, because during the lyophilization process the dye can dry down in different ways, either spread out all over the tube, clumped together, or coating the sides or bottom of the tube. Centrifugation of the tube may not help in collecting the dye solid to the bottom of the tube as this generally works for solutions. However, lyophilized solids are packaged based on highly accurate absorbance measurement of the reagent solution prior to drying, so the vial will contain the correct amount of dye.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

Note that the optimal excitation and emission maxima of AccuBlue® NextGen are 468/507 nm which is different than the other AccuBlue® dyes. The gain settings on your fluorescence plate reader may need to be adjusted for optimal signal to background.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF® Dye succinimidyl ester stability

Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

AccuGreen™ kits are designed for use with the Qubit® fluorometer. All of the other kits are designed for use with 96-well fluorescence plate readers. Our quantitation kits vary in the concentration ranges of dsDNA that they are able to detect. Some of the kits also have different fluorescence excitation/emission. Visit the DNA Quantitation Kit Technology Page or consult the table below to make your selection.

All DNA & RNA Quantitation Kits

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Some products are shipped with blue ice packs as an extra precaution against high temperatures. The blue ice packs may be thawed upon arrival without affecting product performance.

Products with recommended storage at ultra low temperature (-70°C) that also ship on dry ice should arrive frozen. If a product you received was shipped on dry ice and thawed during transit, please contact customer service at order@biotium.com.

You can also download our Product Storage Statement here.

The AccuGreen™ DNA Quantitation Kits are designed for use on the Qubit® fluorometer. The AccuGreen™ High Sensitivity DNA Quantitation Kit is a direct replacement for the Qubit® dsDNA HS Assay Kit from Thermo Fisher Scientific. The AccuGreen™ Broad Range DNA Quantitation Kit is a replacement for the Qubit® dsDNA BR Assay Kit from Thermo Fisher Scientific.

The AccuBlue® Broad Range RNA Quantitation is optimized for use with the Qubit® fluorometer using the pre-programmed RNA Broad Range program. It also can be used with fluorescence microplate readers equipped for detecting far-red fluorescence (Ex/Em 630/660 nm). The kit has a wide linear range of 5-1000 ng RNA, spanning those of both the Quant-iT™ RNA and Quant-iT™ RNA Broad Range Kits from Thermo Fisher Scientific.

The AccuClear® and AccuBlue® DNA Quantitation Kits are designed for use with fluorescence 96-well plate readers. AccuClear®, AccuBlue® NextGen and AccuBlue® High Sensitivity kits require an instrument equipped to read green fluorescence emission (similar to FITC). These assays also can be used with fluorometers such as the Qubit® (Thermo Scientific) and QuantiFluor®-P (Promega). However, due to different linear ranges of the assays, not all of these assays are compatible with the pre-programmed DNA quantitation programs on these instruments. For users who own a Qubit® fluorometer, we recommend using our AccuGreen™ kits, which are designed for use on that instrument.

The AccuBlue® Broad Range Kit requires an instrument equipped to read blue fluorescence (Ex/Em 350/460 nm).

<div> Quant-iT and Qubit are trademarks or registered trademarks of Thermo Fisher Scientific. QuantiFluor is a registered trademark of Promega.</div>

For detection of picogram levels of DNA on a microplate reader, we recommend our AccuBlue® NextGen dsDNA Quantitation Kit (detection range 2.5 pg-3 ng dsDNA) which has higher accuracy and sensitivity than PicoGreen® in a 96-well assay format.

Also see our AccuClear® Ultra High Sensitivity dsDNA Quantation Kit, which is a great all-purpose choice: it has high sensitivity with a broad linear detection range (30 pg-250 ng dsDNA).

The AccuBlue® Broad Range RNA Quantitation has a wide linear range of 5-1000 ng RNA, spanning those of both the Quant-iT™ RNA and Quant-iT™ RNA Broad Range Kits from Thermo Fisher Scientific. It can be used with fluorescence microplate readers equipped for detecting far-red fluorescence (Ex/Em 630/660 nm), or with the Qubit® RNA Broad Range program.

If you own a Qubit® fluorometer, we recommend the following kits, which were designed for use with Qubit®:
AccuGreen™ High Sensitivity dsDNA Quantitation kit is a direct replacement for the Qubit® dsDNA HS Assay kit.
AccuGreen™ Broad Range dsDNA Quantitation Kit is a replacement for the Qubit® dsDNA BR Assay kit.
The AccuBlue® Broad Range RNA Quantitation is optimized for use with the Qubit® fluorometer using the pre-programmed RNA Broad Range program.

Note that the lower limits of kit sensitivity may depend on the detection instrument used.

<div>Quant-iT, Qubit, and PicoGreen are trademarks or registered trademarks of Thermo Fisher Scientific.</div>

A Quantitation Kit contains the quantitation solution components and pre-diluted calf thymus DNA standards. The Quantitation Solution contains the quantitation solution components, and does not include DNA standards.

No. It is recommended to use a DNA standard that most closely resembles your samples. For example, for bacterial DNA quantitation, you may prefer to use lambda DNA as a standard.

Yes, for some of the kits. You can purchase a set of AccuBlue® High Sensitivity standards (cat. no. 31006C) or AccuBlue® Broad Range standards (cat. no. 31007C) separately. We offer a 25 ng/uL DNA standard (cat. no. 31029C) which is the top standard for the AccuClear® Ultra High Sensitivity Assay. And we also offer 1 mg/mL RNase-free DNA (cat. no. 31065).

The dyes are selective for dsDNA; however there is some binding to RNA and ssDNA. Therefore, for the most accurate quantitation, it is recommended to use as clean a dsDNA preparation as possible. Please see the product information sheet for detailed information on RNA and ssDNA binding.

To minimize cost, the trial size kits come with a single DNA standard solution and instructions for preparing a set of standards by dilution. Most of the full size kits are provided with prepared standard sets. The exception is the AccuBlue® NextGen kit, which is supplied with a single standard of 10 ng/uL, with instructions on diluting it to the lower concentrations used in the assay. We found that lower concentrations of DNA were not stable enough for long-term storage.

The AccuBlue® High Sensitivity dye is membrane impermeable to live cells, while PicoGreen® has been shown to readily enter cells. We have not performed such testing with the dyes from our other DNA quantitation kits.

It is highly recommended to minimize cross-over of signals between wells. Depending on your instrument, you can use all black plates or plates with black-walls and clear bottoms.

Some of our DNA quantitation kits contain an enhancer in addition to the DNA quantitation dye. These kits have been optimized with the enhancer to maintain sensitivity and linearity. Omitting the enhancer may affect your results.

Yes. It is recommended to use as clean a dsDNA preparation as possible, but the kits can tolerate some common contaminants. Each kit varies in how it affected by various contaminants. Please see the product information sheets for each kit more information.

The AccuClear®, AccuBlue®, & AccuGreen™ DNA quantitation kits use a high MW dsDNA standard, and are most accurate at quantifying high MW DNA.  While all of the DNA quantitation assays are sensitive to DNA length to some extent, some assays are more accurate with short dsDNA fragments than others.

For quantitation of fragments between 25 and 500 bp:

• AccuBlue® High Sensitivity assay is the most accurate; fluorescence with fragments was ~ 80% of the fluorescence of genomic DNA, and consistent across length and concentration.
• AccuBlue® Broad Range assay is also consistent across length and concentration, with ~ 60% of the fluorescence of genomic DNA.
• AccuGreen™ High Sensitivity assay gives a consistent signal of ~ 80% of the fluorescence of genomic DNA with fragments of 100 bp and higher, but only in the range of 10-100 ng of DNA. Below that, small DNA fragments were inconsistently quantified.
• AccuClear® assay is more variable based on DNA fragment length and concentration. Therefore we do not recommend using this assay with short dsDNA fragments.

To quantify short dsDNA fragments we recommend:

• Use the AccuBlue® High Sensitivity DNA Quantitation assay
• Use sample concentrations well above the stated lower detection limit of the assay (for example, if the stated linear detection range is 0.2-100 ng, for a small fragment you should aim for an amount greater than 2 ng).
• (For best results) Provide your own dsDNA standard of a similar length as your samples.

The assays can be scaled down for 384-well plates. Scale the volumes of all reagents and DNA proportionally. The ratios of DNA, dye, and enhancer (if applicable) should remain the same as in the standard 96-well protocol. However, using less than 200 uL reaction in a 96-well plate may result in lower signal.

Any of our DNA and RNA quantitation kits can be used with the CLARIOstar reader, and we routinely test them on that model. For example, the CLARIOstar gives excellent results with the AccuClear® dsDNA Quantitation Kit, which has the broadest dynamic range, as well as the.AccuBlue® NextGen dsDNA Quantitation Kit, which has the highest sensitivity.

You can find information about our full selection of assays on the dsDNA Quantitation Technology page.

We have tested a variety of different light sources for the development of DNAzure® bands in an agarose gel. We have found that most light sources will work, but how long the development takes is dependent on the brightness of the light. The fastest band development is seen with our Gel-Bright™ and Glo-Plate™ Blue LED illuminators. We have also seen good results with other bright, white LED lights. Other light sources that work but take more time include an LED desk lamp and a cell phone light. We found that a 600W halogen lamp did not work well- it was too hot, which melted the gel.

For optimal staining, we recommend diluting the 100X DNAzure® stock to 1X working solution fresh each time. However, if a 1X solution stored at 4°C and protected from light, the gel stain should to be good up to two weeks.

Yes, the 1X DNAzure® staining solution can be re-used for multiple gels, under certain conditions. Importantly, the staining solution must be removed from the gel before the gel is exposed to light to develop the bands. If the staining solution undergoes the light exposure, it will not be able to stain another gel. The used staining solution should be stored in the refrigerator, protected from light, between uses. We have successfully re-used the staining solution stored for up to 2 weeks, and used up to 4 times with little loss of signal (after 5 or 6 uses, the sensitivity was noticeably lower).

We recommend storing DNAzure® at 4°C. After storage at room temperature, we have seen some loss of dye stability.

EvaGreen® dye is spectrally similar to FAM or SYBR® Green I, which means no change in optical settings is required for using an EvaGreen®-based master mix. However, there are several differences between the dyes.

Lower background fluorescence: EvaGreen® dye has less background than SYBR® Green I due to its novel “release-on-demand” DNA-binding mechanism.

Brighter and non-inhibitory:  EvaGreen® is less inhibitory in the PCR reaction than SYBR® Green, permitting the use of saturating dye concentration for maximal signal and better high-resolution DNA melt analysis.

Safer and more environmentally friendly: EvaGreen® dye is the first and only PCR dye to date designed to be environmentally safe.

Dye stability: EvaGreen® dye is very stable both during storage and under PCR conditions. SYBR® Green I, on the other hand, is known to degrade following multiple freeze-thaw cycles and under PCR conditions. Moreover, decomposed SYBR® Green I is reported to be even more inhibitory to PCR.

Digital PCR: EvaGreen® dye is currently the only qPCR dye to be used in droplet digital PCR (ddPCR).

For most qPCR applications, we recommend using EvaGreen® Dye, 20X  in water (31000), or one of our 2X EvaGreen® qPCR Master Mixes. EvaGreen® Dye, 2000X  in DMSO (31019) is offered for specialized applications requiring a concentrated dye solution.

If necessary, EvaGreen® Dye, 2000X  in DMSO can be diluted in water or master mix for qPCR for a final concentration of 1X in the PCR reaction.

EvaGreen® dye is spectrally similar to FAM or SYBR® Green I, which means no change in optical settings is required for using an EvaGreen®-based master mix compared to SYBR® Green-based master mixes. You can read your signal in the green fluorescence channel (FAM, SYBR® Green, or EvaGreen®) of your instrument.

Biotium offers three different EvaGreen® dye-based qPCR master mixes:

• Forget-Me-Not™ qPCR Master Mix
• Fast EvaGreen® qPCR Master Mix
• Fast Plus EvaGreen® qPCR Master Mix

All three master mixes contain our high-quality EvaGreen® DNA-binding dye and our fast-activating chemically-modified hotstart enzyme, Cheetah™ Taq.

Forget-Me-Not™ qPCR Master Mix is our newest master mix formulation. For most purposes this is the master mix that we would recommend, as it has many benefits: best sensitivity and signal, blue tracking dyes in the master mix and template to reduce pipetting errors, and low cost. Fast EvaGreen® qPCR Master Mix is a sensitive, general use master mix that does not contain blue tracking dyes. Fast Plus EvaGreen® qPCR Master Mix has a slightly different formulation than the regular Fast EvaGreen® qPCR Master Mix, and was designed for more challenging samples.

Forget-Me-Not™ EvaGreen® qPCR Master Mix is a hot-start EvaGreen® dye-based master mix. It uses Biotium’s proprietary chemically modified hot-start DNA Polymerase, Cheetah™ Taq. This master mix also contains a light blue tracking dye, and comes with a dark blue template dye, for two-color tracking to reduce pipetting errors. The user only needs to supply primers and template.

Forget-Me-Not™ Universal Probe Master Mix is formulated for fluorescent probe-based PCR applications. It uses Biotium’s proprietary chemically modified hot-start DNA Polymerase, Cheetah™ Taq. The user needs to supply some kind of fluorescent probe (such as TaqMan®, dual-labeled BHQ®, or Molecular Beacon) in addition to primers and template.

Both of these qPCR master mixes contain Cheetah™ Taq (our chemically modified hotstart DNA polymerase), buffer and dNTPs.

The Fast EvaGreen® Master Mixes also contain the DNA-binding dye EvaGreen®, and are used for fluorescent dye-based qPCR. The only thing the user needs to supply are primers and template.

The Fast Probe Master Mix  is formulated for conducting real-time PCR using an oligo-based probe, such as a TaqMan™ probe, an MGB probe or an AllGlo™ probe. The user needs to supply primers, probe and template.

The ROX concentration is 35 nM which is suitable for low ROX instruments. If you are using a high ROX instrument, you can add ROX, 25 mM in TE Buffer (Catalog # 29052) which is available separately (Cat # 29052).

EvaGreen® Dye is supplied at 20X (25 uM) in water (cat. no. 31000) or 2000X (2.5 mM) in DMSO (cat. no. 31019). Other formulations are available for product licensees, inquire at btinfo@biotium.com.

Also see our ready-to-use EvaGreen® Master Mixes for qPCR.

Our original EvaGreen® Dye is a saturating dsDNA binding dye that is superior for quantitative real-time PCR (qPCR), high-resolution melt (HRM), digital droplet PCR (ddPCR) and other genomics applications over SYBR® Green I and other commercial PCR dyes. It offers several essential features critical for PCR and related applications including high thermal, chemical and photostability, high sensitivity due to high signal to noise related to its novel ‘release-on-demand’ mechanism, non-inhibitory to PCR, and lack of dye migration.  In addition, EvaGreen® Dye is non-toxic, non-mutagenic, and not hazardous to aquatic life.

EvaGreen® Plus Dye is an advanced version of the original EvaGreen® Dye, retaining the same essential benefits while providing a higher signal-to-noise ratio. This greater sensitivity offers further advantages for digital PCR and isothermal applications.

EvaGreen® Dye is used in all of Biotium’s dye-based qPCR master mixes, which combine superior brightness and sensitivity, with the ability to do melt curve analysis in the same reaction. The new EvaGreen® Plus Dye is available as a stand-alone 20X solution in water.

Our qPCR Master Mixes are all offered with optional ROX reference dye, which is used by some qPCR instruments to normalize the PCR fluorescence. We also include instructions for using no ROX, low ROX, or high ROX, depending on the instrument you are using. Based on instrument manufacturers’ recommendations, we recommend the following:

No ROX

BioRad: iCycler™, MyiQ™, MiQ™2, iQ™5, CFX-96 Touch™, CFX-384 Touch™, Chromo4™, MiniOpticon™

Qiagen: Rotor-Gene® Q, Rotor-Gene® 3000, Rotor-Gene® 6000

Eppendorf: Mastercycler® Realplex

Illumina: Eco™ RealTime PCR System

Cepheid: SmartCycler®

Roche: LightCycler® 480, LightCycler® 2.0

Low ROX

ABI: 7500, 7500 Fast, ViiA 7™, QuantStudio™

Stratagene: MX4000P, MX3000P, MX3005P

High ROX

ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne®, StepOne Plus®

0.5X to 1X EvaGreen® is commonly used for LAMP applications when fluorescent signal is read by instrumentation. For detection by eye, an increased concentration of EvaGreen® can be used. For example,  Lee, S. H. et al. tested up to 100X EvaGreen® and found that 10X EvaGreen® was optimal for their assay.

Lee, S. H. et al. Front. Microbiol., 09 January 2017 | https://doi.org/10.3389/fmicb.2016.02166

EverBrite™ Mounting Medium (23001) and EverBrite™ Mounting Medium with DAPI (23002) both have a refractive index of 1.46.  EverBrite™ Hardset Mounting Medium (23003) and EverBrite™ Hardset Mounting Medium with DAPI (23004) both have an initial refractive index of 1.38, which increases to 1.42 after 24 hours of curing, and 1.46 after 4 days, after which it remains constant

Yes, EverBrite™ mounting media are compatible with a wide range of dyes. Unlike VectaShield®, EverBrite™ won’t interfere with cyanine-based fluorophores like Alexa Fluor® 647, Cy®3, or Cy®5.

AlexaFluor is a registered trademark of Thermo Fisher Scientific; Cy Dye is a registered trademark of Cytiva; VectaShield is a registered trademark of Vector Laboratories.

EverBrite™ wet-set mounting medium is an aqueous, glycerol-containing medium. It can be removed from samples by washing several times with PBS or other buffer. However, washing with PBS will not remove DAPI staining.

For coverslips mounted on slides with EverBrite™ Hardset or EverBrite™ wet-set medium, the slides can be soaked in PBS in a slide jar for several hours with gentle agitation until the coverslip slides off (avoid prying or pulling the coverslip, which could damage the sample). Then wash the slide several times to remove any remaining mounting medium. If the coverslips have been sealed with CoverGrip™ Coverslip Sealant, soaking in PBS will remove the sealant as well. Other methods may be required to remove other types of coverslip sealants.

CoverGrip™ is designed to replace nail polish for permanent sealing of wet-mounted coverslips. It can be removed by soaking slides in buffer (see the product information sheet for details). CoverGrip™ is not designed to create a removable temporary seal for hybridization.

EverBrite™ mounting medium does show high background fluorescence with violet excitation. The background is more prominent when imaged using epifluorescence using UV excitation and a DAPI filter cube, but is generally quite low in confocal imaging using 405 nm laser.

CellBrite® Cytoplasmic Membrane Dyes do not efficiently stain EVs. Some of the CellBrite® Fix, MemBrite® Fix, and CellBrite® Steady dye options can be used for this application. However, for optimal staining of exosome membranes we recommend our ExoBrite™ True EV Membrane Stains, which are novel lipophilic membrane dyes specifically designed and optimized for efficient staining of EV membranes with minimal dye aggregation. See our Extracellular Vesicle Research page for more information about our complete line of EV stains and antibodies

While early studies of EVs attempted to use first-generation membrane dyes like DiI or PKH to stain EVs, more recently this class of dyes has been found to be largely unsuitable for EV staining due to their high degree of aggregation. Dye aggregation not only generates nonspecific particles that are indistinguishable from EVs in flow cytometry, but also results in poor EV labeling efficiency. Biotium developed the ExoBrite™ True EV Membrane Stains in response to our customers’ difficulties with using traditional membrane dyes to stain EVs. See our Literature Digest for more information.

Yes, EVs can be stained simultaneously with an ExoBrite™ True EV Membrane Stain and a fluorescent antibody.

With purified EVs, we have seen good results when EVs were stained in 500 mL of 1X ExoBrite™ plus 1 ug/mL fluorescent antibody. Please view our Product Information Sheet for detailed protocols.

We strongly recommend our ExoBrite™ Flow Antibody Conjugates for staining both purified or bead-bound EVs. The antibodies are validated and optimized to offer bright signal and low background. They are available against human or mouse CD9, CD63, and CD81 tetraspanin proteins.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

The main difference between GelRed® and GelGreen® is their fluorescence excitation and emission wavelengths. GelRed® has red fluorescence, similar to ethidium bromide. GelGreen® has green fluorescence, similar to SYBR® Green or SYBR® Safe. Both dyes are compatible with standard UV transilluminators. GelGreen® is also compatible with blue light transilluminators, which allow users to avoid exposing themselves and their DNA samples to ultraviolet radiation.

GelRed® and GelGreen® have higher sensitivity for double stranded nucleic acids compared to single stranded nucleic acids, but GelRed® is more sensitive for staining single stranded nucleic acids than GelGreen®. GelRed® is about twice as sensitive for double stranded nucleic acids compared to single-stranded nucleic acids, and about five times more sensitive than GelGreen® for staining single stranded nucleic acids.

For more information about these products, please visit our DNA stains technology page.

View the GelRed® Product Line

View the GelGreen® Product Line

The water formulation is a newer and improved product compared to the stock in DMSO. We recommend using dyes in water to avoid the potential hazards of handling DMSO, which can be absorbed through the skin. We continue to offer dyes in DMSO because some users do not wish to alter their established laboratory protocols. Based on internal testing, both formulations perform similarly.

GelRed® and GelGreen® can be added to agarose during gel casting at a final concentration of 1X, or they can be used for post-electrophoresis gel staining at a final concentration of 3X in water. For detailed protocols for use, please download the GelRed® Product Information Sheet or GelGreen® Product Information Sheet.

Many customers use GelRed® or GelGreen® precast gels for convenience. However, because GelRed® and GelGreen® are high affinity dyes designed to be larger dyes to improve their safety, they can affect the migration of DNA in precast gels. Some samples, such as restriction digested DNA may migrate abnormally in GelRed® or GelGreen® precast gels.

Tip #1: Load less DNA

Smearing and smiling in GelRed® or GelGreen® precast gels most often caused by overloading of DNA. If you see band migration shifts or smearing and smiling, try reducing the amount of DNA loaded. The recommended loading amount for ladders and samples of known concentration is 50-200 ng/lane. For samples of unknown concentration, try loading one half or one third of the usual amount of DNA. This usually solves band migration problems.

Tip #2: Try the post-staining protocol

To avoid any interference the dye may have on DNA migration, we recommend using the post-staining protocol. If your application requires loading more than the recommended amount of DNA, use the post-staining protocol. While we recommend post-staining gels for 30 minutes, you may be able see bands in as little as five minutes, depending on how much DNA is present. Post-staining solutions can be reused. See the GelRed® Product Information Sheet or GelGreen® Product Information Sheet for detailed protocols.

Other tips to improve agarose gel resolution:

• If you see DNA migration issues or smearing after post-staining with GelRed® or GelGreen®, then the problem is not caused by the nucleic acid dye. Avoid overfilling gel wells to prevent smearing of DNA down the surface of the gel.
• Pour a lower percentage agarose gel. Higher molecular weight DNA separates better with a lower percentage gel.
• Change the running buffer. TBE buffer has a higher buffering capacity than TAE buffer.

There are a few possibilities:

1. The dye may have precipitated out of solution.
   • Heat the GelRed® or GelGreen® solution to 45-50°C for two minutes and vortex to dissolve.
   • Store dye at room temperature to avoid precipitation.
2. If you are seeing high background staining of the gel, the agarose that you are using may be of low quality. Contaminants in the agarose may bind to the dye, resulting in increased background.

We have tested a variety of DNA ladders from different suppliers with GelRed® and GelGreen® with good results. Similarly, any common DNA loading buffers can be used. Band migration problems or smearing or smiling DNA bands are most commonly caused by overloading of ladder. If you are experiencing band migration problems with your ladder, please try reducing the amount of ladder you load. We recommend loading 50-200 ng ladder per lane for GelRed® or GelGreen® precast gels. See the FAQ for smearing or smiling DNA bands for more tips.

Biotium also offers 1 kb and 100 bp DNA ladders in TE buffer or ready-to-use format in loading buffer. Ladders are supplied with a separate tube of 6X DNA Loading Buffer to use with your samples.

GelRed® and GelGreen® are stable dyes. You can use the dyes in room light. However, we recommend storage of the dyes in the dark between uses. We have had a customer report using GelRed® with success after accidentally leaving it in ambient light for one month.

GelRed® and GelGreen® can be used to stain both ssDNA and RNA, but GelRed® is about 5 times more sensitive for single-stranded nucleic acids than GelGreen®. Titration assays using a fluorescence microplate reader showed that the fluorescence signal of GelRed® bound to ssDNA and RNA is about half that of GelRed® bound to dsDNA.

We and other users have often observed that GelGreen® stains ssDNA and RNA orange/ pink and dsDNA green. We have also seen that smaller dsDNA fragments can appear orange-pink, the color ranging from white-pink-orange. We are not sure about the underlying mechanism, possibly the structure of single-stranded nucleic acids favors an altered stacking interaction of GelGreen® monomers leading to the formation of J-aggregates that have red emission.

Yes, customers have reported using GelRed® in glyoxal and formaldehyde agarose gels for precast staining of RNA.

Yes, see Ana. Biochem. doi: http://dx.doi.org/10.1016/j.ab.2012.09.003 .

Yes, use the post-staining protocol for polyacrylamide gels. For polyacrylamide gels containing 3.5-10% acrylamide, typical staining time is 30 minutes to 1 hour with gels of higher acrylamide content requiring longer staining time.,

Biotium also offers PAGE GelRed® a non-toxic, non-mutagenic dye specifically designed for staining DNA in polyacrylamide gels.

Yes. Please use the post-staining procedure for DGGE and EMSA gels. For PFGE gels, the pre-cast or post-staining protocol may be used.

Yes, GelRed® is compatible with alkaline running buffer.

Yes, GelRed® and GelGreen® can be used for Comet assay.

Yes, see Virol J. 2012, 9, 110.

Yes, see Electrophoresis, doi: 10.1002/elps.201200624 .

Customers have reported using GelRed® in cesium gradients. To extract GelRed® from DNA after cesium banding, we recommend adding SDS to a final concentration of 0.1% before butanol extraction. Alternatively, chloroform can be used instead of butanol for extraction.

GelRed® and GelGreen® are ultra-sensitive dyes that require loading only 1/2 to 1/3 the sample amount that you would use for ethidium bromide (EtBr). However, the sensitivity of the staining will depend on the instrument capability and exposure settings. For maximum sensitivity, we recommend our EMBER™ Ultra DNA Gel Kit which can detect as little as ≤1 ng DNA.

GelRed® has been shown to bind DNA exclusively by intercalation (https://link.springer.com/article/10.1007/s00249-014-0995-4). GelGreen® most likely binds by a combination of intercalation and electrostatic interaction.

GelRed® and GelGreen® do not migrate through the gel as easily as ethidium bromide. It is not necessary to add additional dye to the running buffer, and the gel will be stained more homogenously than a gel stained with ethidium bromide.

Yes. We recommend Qiagen or Zymoclean™ gel extraction kits or phenol-chloroform extraction to remove the dye from DNA. Some users have reported performing PCR on DNA in the presence of GelRed® with no purification step, for example by incubating GelRed®-stained gel slices in TE buffer to extract DNA by passive diffusion for use in PCR, or by using a few microliters of molten agarose from GelRed®-stained gel slices containing DNA for PCR.

We recommend our DNA Gel extraction kit to remove the GelRed® from a DNA sample, but some customers report success in removing GelRed® by phenol-chloroform extraction.

Customers also report good results using ZymoClean™ Gel DNA Recovery Kit from Zymo Research, GenElute™ Agarose Spin Column from Sigma, PureLink® Quick Gel Extraction kit from Life Technologies, illustra GFX PCR DNA and Gel Band Purification kit from GE Healthcare, High Pure PCR Product Purification Kit from Roche Applied Sciences, and GenJET gel extraction kit from Thermo Scientific.

GelRed® has been validated for Southern blotting. We recommend using the post-staining protocol for blotting applications.

GelRed® and GelGreen® can be added to agarose during gel casting at a final concentration of 1X, or used for post-electrophoresis gel staining at a final concentration of 3X in water. Dilute the 10,000X stock 10,000-fold for 1X precast gels (for example, 5 uL for a 50 mL gel), or 3,333-fold for a 3X post staining solution (15 uL for a 50 mL solution).

A 0.5 mL vial of GelRed® or GelGreen® is can be used to prepare 100 minigels (50 mL each) using the precast protocol, or for post-electrophoresis staining of 33 minigels in 50 mL staining solution per gel. Post-staining solution also can be re-used for staining two or more gels. For detailed protocols for use, please download the GelRed® Product Information Sheet or GelGreen® Product Information Sheet.

No, but de-staining with water can be performed if background is high.

Yes. However, if the sensitivity decreases, use a fresh solution of the dyes.

We don’t recommend adding GelRed® or GelGreen® directly to loading buffer, because this can result in inaccurate band migration. Biotium offers 6X GelRed® Prestain Loading Buffers designed for this application, although we do not recommended them for applications where precise DNA band sizing is required. For the most accurate determination of DNA band sizes, we recommend using GelRed® post-staining (see the GelRed® protocol for details).

GelRed® is compatible with a standard UV transilluminator (302 or 312 nm).
GelGreen® has sufficient absorption between 250-300 nm and a strong absorption peak at around 500 nm. GelGreen® is compatible with a 254 nm UV transilluminator or a gel reader with visible light excitation such as a Dark Reader or a 488 nm laser gel scanner.

Use the ethidium bromide filter for GelRed®; use a SYBR® Green or yellow filter for GelGreen®. Alternatively, a long-pass yellow filter can be used with both GelRed® and GelGreen®. Please review the emission spectra for GelRed® and GelGreen® for more specific wavelengths.

You can store precast GelRed® gels for up to a week, and GelGreen® gels for up to a month. We recommend storing gels at room temperature in the dark. We no longer recommend storing gels at 4°C, because this can lead to dye precipitation and poor performance.

No. We do not recommend that GelRed®/GelGreen® gels be reused after electrophoresis because the staining intensity can be decreased with sequential electrophoresis.

Yes, unused solidified agarose with GelRed® or GelGreen® can be remelted. If the unused agarose with dye is to be stored for more than a day or so, we recommend protecting it from light.

GelRed® is more stable than GelGreen®. We do not recommend storing GelRed® in molten agarose for more than a few days.

In AMES and related tests, GelRed® and GelGreen® were shown to be much safer alternatives to EtBr and SYBR dyes. Nevertheless, please exercise safe laboratory practices when using these reagents.

Please visit our website www.biotium.com to download a comprehensive safety report.

GelRed® and GelGreen® is classified as non-hazardous for drain disposal under California Code of Regulations Title 22. Some facilities have approved the disposal of GelRed® and GelGreen® directly down the drain. However, because regulations vary, please contact your safety office for local disposal guidelines. Please review the GelRed®/GelGreen® safety report for more detailed information.

Many customers use GelRed® or GelGreen® precast gels for convenience. However, because GelRed® and GelGreen® are high affinity dyes designed to be larger dyes to improve their safety, they can affect the migration of DNA in precast gels. Some samples, such as restriction digested DNA may migrate abnormally in GelRed® or GelGreen® precast gels.

Tip #1: Load less DNA

Smearing and smiling in GelRed® or GelGreen® precast gels most often caused by overloading of DNA. If you see band migration shifts or smearing and smiling, try reducing the amount of DNA loaded. The recommended loading amount for ladders and samples of known concentration is 50-200 ng/lane. For samples of unknown concentration, try loading one half or one third of the usual amount of DNA. This usually solves band migration problems.

Tip #2: Try the post-staining protocol

To avoid any interference the dye may have on DNA migration, we recommend using the post-staining protocol. If your application requires loading more than the recommended amount of DNA, use the post-staining protocol. While we recommend post-staining gels for 30 minutes, you may be able see bands in as little as five minutes, depending on how much DNA is present. Post-staining solutions can be reused. See the GelRed® Product Information Sheet or GelGreen® Product Information Sheet for detailed protocols.

Other tips to improve agarose gel resolution:

• If you see DNA migration issues or smearing after post-staining with GelRed® or GelGreen®, then the problem is not caused by the nucleic acid dye. Avoid overfilling gel wells to prevent smearing of DNA down the surface of the gel.
• Pour a lower percentage agarose gel. Higher molecular weight DNA separates better with a lower percentage gel.
• Change the running buffer. TBE buffer has a higher buffering capacity than TAE buffer.

There are a few possibilities:

1. The dye may have precipitated out of solution.
   • Heat the GelRed® or GelGreen® solution to 45-50°C for two minutes and vortex to dissolve.
   • Store dye at room temperature to avoid precipitation.
2. If you are seeing high background staining of the gel, the agarose that you are using may be of low quality. Contaminants in the agarose may bind to the dye, resulting in increased background.

BAPTA-based calcium indicators and chelators have been shown to be fixable in situ by EDC (EDAC)¹, #59002 which would be compatible with subsequent immunofluorescence and IHC.

¹Cell Calcium. 1997. 21(3), 175; DOI:10.1016/S0143-4160(97)90042-7.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

Some of Biotium’s luciferase assay kits can be used as alternatives to Promega kits. The comparable products are listed below. Click on a link for product information.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF® Dye succinimidyl ester stability

Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Some products are shipped with blue ice packs as an extra precaution against high temperatures. The blue ice packs may be thawed upon arrival without affecting product performance.

Products with recommended storage at ultra low temperature (-70°C) that also ship on dry ice should arrive frozen. If a product you received was shipped on dry ice and thawed during transit, please contact customer service at order@biotium.com.

You can also download our Product Storage Statement here.

No. The buffers are based on different proprietary formulations.

The Passive Lysis Buffer (Renilla) is compatible with the Firefly Assay, but the Firefly Lysis Buffer is not compatible with the Renilla Assay.

Yes, please check the website for catalog numbers and pricing.

No. Biotium’s Renilla and Firefly Assay Kits are based on flash luminescence. Samples should be treated and read individually. Alternatively, you may purchase the Steady-Glo Firefly Assay Kit for HTS. We currently do not have a Steady-Glo for Renilla Assays.

Renilla is often used as an internal control for transfection efficiency while the Firefly luciferase vector is used for reporter activity. They also can be reversed, with Renilla as the reporter and Firefly as the internal control.

We haven’t done a side-by-side comparison of the One-Glo™ and Steady-Luc™ assays, but based on the comparison of One-Glo and Steady-Glo on Promega’s website, we expect the signal from Steady-Luc™ assay to similar to that of the Steady-Glo™.

Our Steady-Luc™ reagent is a homogenous luciferase assay that can be added directly to culture medium like One-Glo™ reagent, and the Steady-Luc™ working solution is stable for at least 18 hours at RT.

No, our new Firefly & Renilla Single Tube Assay Kit allows you to measure firefly and Renilla luciferase activities sequentially in a single sample of lysate.

Assay sensitivity is dependent on many factors including the promoter strength of each vector, the transfection efficiency of each vector, and the cell type used. In general, Firefly luciferase is more widely used because it has a longer half-life and and better stability than Renilla luciferase.

Biotium’s Renilla and Firefly Assays are flash luminescence assays. The approximate half-lives are 1-2 minutes for Renilla and 10 minutes for Firefly. The Steady-Glo Firefly Assay has a half-life of about 3-5 hrs. However, the signal is about 1/10 of the flash luminescence assays.

Luminescence values are relative and depend on the experimental system. For example, luminometers from two different manufacturers each may have 6-log capability, but one may measure a range of 1 to 1,000,000 relative light units (RLU) while the other measures 0.01 to 10,000 RLU. Both instruments can measure a dynamic range of 6-log units, but they read out different arbitrary units.

The firefly luciferase enzyme provided in the ATP-Glo™ kit is recombinant, produced in E. coli.

Coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

To date, we have not identified a fluorescent cellular stain that will detect bacteria but not mammalian cells with high specificity, or vice versa. While some mammalian cell stains show weak staining of bacteria, they usually do show some signal, and will frequently stain dead bacteria more intensely than live bacteria.

We offer a selection of antibodies for specific bacterial antigens, which potentially have applications for differential staining of bacteria vs. mammalian cells, but we have not validated them in co-culture models.

Also see our Viability PCR Technology Page to learn about how PMA dye can be used for highly specific detection of microbial cell viability in complex samples.

Fixation is not recommended after staining with the Viability/Cytotoxicity Assay Kit (cat. # 30027) as the dead cell dye, EthD-III will transfer to all cells and not maintain dead cell-specific staining. Our Live-or-Dye™ NucFix™ Red (cat. # 32010) is a formaldehyde-fixable dead cell dye that can be used in bacteria.

Viability qPCR (vPCR) can be an alternative strategy for quantitating live and dead bacteria. Using dead cell-specific dyes, PMA (cat. # 40019) or PMAxx (cat. # 40069) that covalently modify DNA (of only dead cells) after photo-crosslinking, a simple quantitative PCR (qPCR) amplification is used to selectively amplify live-cell DNA. Learn more about the vPCR technology.

CellBrite® and MemBrite® Stains were originally developed for staining mammalian cells in culture, but some of the stains also have been validated for other organisms and applications. For dyes to stain yeast or bacteria membranes, see Cellular Stains in Different Organisms. For information on staining other organisms or cell types, please see our Tech Tip: Researching Applications for Membrane Dyes.

The CellBrite® Cytoplasmic Membrane Dyes do not stain bacteria. The reactive CellBrite® Fix dyes stain both gram-positive and gram-negative bacteria, while the MemBrite® Fix dyes stain only gram-positive bacteria. However we have not tested these dyes for cell division tracking in bacteria.

There is literature describing the use of CFSE to track bacterial cell division,  the ViaFluor® SE cell proliferation dyes are likely to work in a similar manner, but we have not tested this.

See our Cellular Stains Table for a comprehensive list of cellular stains with their ability to stain various cell types.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF® Dye succinimidyl ester stability

Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

See our CF® Dye Quick Reference Table for a list of dyes and summary of their features. Our CF® Dye Selection Guide has more detailed information on each CF® dye, and ordering information for our various CF® dye product lines. You can download CF® dye normalized absorption and emission data in Excel format.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Some products are shipped with blue ice packs as an extra precaution against high temperatures. The blue ice packs may be thawed upon arrival without affecting product performance.

Products with recommended storage at ultra low temperature (-70°C) that also ship on dry ice should arrive frozen. If a product you received was shipped on dry ice and thawed during transit, please contact customer service at order@biotium.com.

You can also download our Product Storage Statement here.

For CF® dye, cyanine dye, FITC, biotin, DNP, and dig kits, see the Mix-n-Stain™ Kit Compatibility and Protocol Selection Flowchart, below. We also recommend downloading the updated Product Protocol and completing the pre-labeling checklist to find the right protocol and kit size to use for each antibody you wish to label. Learn more about CF® dyes on our CF® Dye Technology Page, or download the CF® Dye Brochure.

Note that Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols. Check the protocol for your kit to verify that your antibody is compatible with labeling, then choose a kit size that matches your the amount of antibody you wish to label.

CF® Dyes are highly water soluble, small organic dyes designed by scientists at Biotium for labeling proteins and nucleic acids. With a series of over 35 colors (and growing), many of our CF® Dyes are brighter and more photostable than competing dyes. For more information please see the product flyers for individual CF® Dyes, the CF® Dye Selection Guide, and our CF® Dye FAQs.

PBS, HEPES, MES, MOPS or borate buffers are compatible. Buffers containing up to 20 mM Tris are also compatible. Tris at levels higher than 20 mM should be removed using the ultrafiltration vial provided in the kit. See the Product Protocol for more information.

While labeling antibody without stabilizer proteins generally gives the best results, but antibodies with BSA or gelatin can be labeled with Mix-n-Stain™ CF® Dye Antibody Labeling Kits. The BSA or gelatin will also be labeled, but due to the high solubility of CF® dyes, the labeled BSA or gelatin is washed away during standard immunofluorescence staining. We recommend downloading the updated Product Protocol and completing the pre-labeling checklist to find the right protocol and kit size to use for each antibody you wish to label. Also see the Mix-n-Stain™ Kit Compatibility and Protocol Selection Flowchart, below.

Note that Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols. Check the protocol for your kit to verify that your antibody is compatible with labeling, then choose a kit size that matches your the amount of antibody you wish to label.

IgG in ascites fluid can be labeled with Mix-n-Stain™ CF® Dye Antibody Labeling Kits with good results using a larger sized kit and our modified Mix-n-Stain™ protocol. Download the updated Product Protocol and complete the pre-labeling checklist to select the appropriate kit size and labeling protocol.

Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols, and cannot be used to label antibodies in ascites fluid. Check the protocol for your kit to verify that your antibody is compatible with labeling, then choose a kit size that matches your the amount of antibody you wish to label.

Mix-n-Stain™ labeling does not work well for antibodies in serum or hybridoma cell culture supernatant. We recommend purifying IgG from serum or supernatant before labeling.

For CF® dye, cyanine dye, FITC, biotin, DNP, and dig Mix-n-Stain™ kits, sodium azide, EDTA, small sugars, and <10% glycerol have no effect on the labeling. Higher levels of glycerol, or any level of DTT, 2-mercaptoethanol or free amino acids (such as glycine) should be removed using the ultrafiltration vial provided in the kit. See the Product Protocol for more information.

Note that Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols. Check the protocol for your kit to verify that your antibody is compatible with labeling, then choose a kit size that matches your the amount of antibody you wish to label.

Mix-n-Stain™ labeling results in covalent linkage of dye and antibody, so there will be no dye diffusion or transfer.

Direct immunofluorescence staining eliminates the need for secondary antibody incubation and wash steps, and allows the use of multiple primary antibodies from the same species for multicolor detection, or staining of animal tissues with antibodies raised in the same species without secondary antibody cross-reactivity (e.g. mouse-on-mouse staining).

1. Unlike Zenon, Mix-n-Stain™ labeling covalently attaches the dye to the antibody, eliminating dye transfer or diffusion between antibodies during multi-color staining.
2. Mix-n-Stain™ conjugates are stable for at least 6 months in storage buffer, whereas Zenon complexes must be used within 30 minutes.
3. Mix-n-Stain™ conjugates are less bulky because the dyes are directly linked to the antibody, unlike Zenon conjugates which use antibody fragments.
4. No post-staining fixation is required with Mix-n-Stain™.
5. Unlike Zenon, Mix-n-Stain labeling is not species-specific.

The main advantage of Mix-n-Stain™ antibody labeling kits is that they include our novel CF® dyes, which are brighter and more photostable than the dyes provided in Lightning Link® kits. Mix-n-Stain™ kits are sized for labeling smaller amounts of antibody and are sold as single labelings, providing more flexibility compared to Lightning Link® kits.

The dye is covalently attached to the side chains of amino acids located far away from the antigen-binding sites so that the affinity of the antibody is not affected. However, the exact attachment sites and the nature of chemical linkages are proprietary information.

The Mix-n-Stain™ kits do not specifically label the Fc region of the antibody, they will label accessible reactive residues irrespective of the location on the antibody. The image of the labeled dye on Fc region of antibody is merely representative. We design our Mix-n-Stain™ labeling kits to give the optimal DOL for each dye to minimize any effect on the binding affinity of the antibody. Based on this, it is likely that the majority of the labeling is on the Fc region of the antibody, which does not directly bind antigen. There are some cases where antibody labeling can disrupt antibody-antigen binding, usually for monoclonal antibodies, but this is rare based on our experience.

The degree of labeling with Mix-n-Stain™ kits is estimated to be in the range of 4-6 dye molecules per antibody.

This question relates to a key element of our invention. The unique formulations of our dyes and buffers and the labeling strategy have completely removed this concern, which normally has to be dealt with when using conventional antibody labeling methodology. The exact mechanism on how this problem is solved is proprietary information. For use on live cells we do recommend a quick purification using the provided ultrafiltration vial or staining at 4°C to prevent possible endocytosis of free dye.

There is no need to measure the dye amount or vary the reaction time as long as the amount of your antibody to be labeled falls within the range specified for each kit. With Mix-n-Stain™ labeling kits optimal labeling is ensured because of the proprietary dyes and reaction buffer.

The kits are optimized for labeling antibodies with a concentration between 0.5-1.0 mg/mL. If your antibody solution is too dilute, you can concentrate it by centrifugation using the ultra-filtration vial provided in the kit. If your antibody solution is too concentrated, you can dilute it with 1x PBS. Antibody concentrations outside the recommended range may result in either under or over labeling.

Approximately 100%.

For the Mix-n-Stain™ CF® Dye, biotin, Cyanine Dye, FITC, and hapten antibody labeling kits it takes 30 seconds or less to mix the components (antibody, dye and reaction buffer). After another 15 minutes of hands-free reaction time, you are done! You can use the antibody right away to stain your sample for microscopy, flow cytometry, Western analysis, or other applications requiring fluorescently labeled antibodies.

The labeling reaction in the Mix-n-Stain™ CF® Dye, biotin, Cyanine Dye, FITC, and hapten antibody labeling kits will be complete after 15 minutes. However, a longer reaction time will not adversely affect the labeling.

No. The Mix-n-Stain™ kits are optimized for 1 labeling. We do not recommend trying to split the kit to label more than one antibody or for more than one use.

Your labeled antibody is stable for at least six months if stored in the Mix-n-Stain™ storage buffer at 4°C . For longer-term storage, you can aliquot your antibody and store at -20°C.

We now offer Mix-n-Stain™ Nanobody Labeling Kits, specifically designed for optimized labeling of single-domain nanobodies with CF® dyes.

We also have had customers report successful labeling of nanobodies with our original CF® Dye Mix-n-Stain™ Antibody Labeling Kits. Mix-n-Stain™ kits are optimized for labeling whole IgG (150 kDa) with 4-6 dyes per antibody. Due to their smaller size (~15 kDa), single chain antibodies have fewer potential labeling sites. So to increase the degree of labeling for single chain antibodies, we recommend using a higher ratio of dye to protein. For example, the Mix-n-Stain™ 50-100 ug size kit could be used to label 25-30 ug of nanobody. Also, we do not recommend using the 10K MWCO ultrafiltration vial with proteins smaller than 30 kDa. If ultrafiltration is required prior to Mix-n-Stain™ labeling, a 3K MWCO ultrafiltration vial (catalog no. 22018) should be used instead.

Mix-n-Stain™ kits are optimized for labeling IgG antibodies, but can be used to label other proteins.

Customers have reported successful labeling of nanobodies and single chain antibodies. We now offer Mix-n-Stain™ Nanobody Labeling Kits, specifically designed for optimal labeling of nanobodies with CF® dyes.

Mix-n-Stain™ kits labeling conditions may cause denaturation of IgM antibodies. There are also published reports of Mix-n-Stain™ labeling of enzymes and lectins. Note that any conjugation method, including Mix-n-Stain™, may affect the biological activity of proteins. Also, some free unreactive dye may remain after Mix-n-Stain™ labeling, which could interfere with live cell staining or trafficking studies using fluorescently labeled proteins. The ultrafiltration vial provided in the kit can be used to remove free dye after labeling if necessary.

Also see our CF® Dye SE Protein Labeling Kits, which include everything you need to label and purify up to 3 mg protein. We also offer VivoBrite™ Antibody Labeling Kits for labeling antibodies or other proteins with near-infrared CF® dyes for small animal in vivo imaging.

Our Mix-n-Stain™ kits are designed for labeling antibodies and would not be suitable for labeling oligos. To label oligos with CF® Dyes, please see FAQ Can CF® Dyes be used to label oligos?

Currently, we do not have any products for conjugating DNA to HRP or other enzymes. However, you can order oligos pre-conjugated to HRP from oligo suppliers.

Yes, Mix-n-Stain™ kits can be used to label antibodies containing the preservative ProClin™ 300.

Direct immunofluorescence detection can be less sensitive than indirect detection. You may need to use a higher concentration of antibody or higher gain settings to achieve similar staining intensity compared to indirect immunofluorescence staining. In our internal testing, indirect immunofluorescence staining results in about 3-fold signal amplification compared to direct immunofluorescence staining.

Mix-n-Stain™ kits contain a very small quantity of dye or biotin, which usually is not visible in dry form. Once you add your antibody solution to the vial and mix, the solution will become brightly colored. Biotin, CF™350, CF™405M, and CF™405S Mix-n-Stain™ compounds yield colorless or very pale solutions, but rest assured that the vial does contain the dye or biotin.

For CF® Dye, biotin, dig, or DNP  kits, please download the updated Product Protocol and follow the Troubleshooting Checklist for step-by-step troubleshooting of Mix-n-Stain™ labeling and subsequent staining.

Note that Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols. Check the protocol for your kit to verify that your antibody is compatible with labeling, then contact technical support for assistance.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF® Dye succinimidyl ester stability

Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Some products are shipped with blue ice packs as an extra precaution against high temperatures. The blue ice packs may be thawed upon arrival without affecting product performance.

Products with recommended storage at ultra low temperature (-70°C) that also ship on dry ice should arrive frozen. If a product you received was shipped on dry ice and thawed during transit, please contact customer service at order@biotium.com.

You can also download our Product Storage Statement here.

Like other caspase-3 substrates, NucView® 488 Caspase-3 Substrate is based on a DEVD sequence that also can be cleaved by caspase-7.

Click here to view a list of primary cell types and immortalized cell lines reported to work with NucView® Caspase-3 Substrates.

The substrates are very stable. Some users have reported performing time course assays with NucView® 488 Caspase-3 Substrate for 4-5 days.

The NucView® caspase-3 substrates are activity-dependent i.e. require active caspase-3 enzyme. In dead, fixed and preserved cells or tissue sections, there are no active caspases and hence these substrates cannot be used. We also offer TUNEL assay kits that are suitable for apoptosis detection in fixed cells and tissues. The kits employ dUTPs conjugated to our exceptionally bright and photostable CF® dyes for single-step fluorescent TUNEL labeling of DNA strand breaks, a hallmark of apoptotic cells, and are suitable for analysis by fluorescence microscopy or flow cytometry.

NucView® Caspase-3 Substrates can be added to the cells at the start of the experiment or at the end. A major advantage of NucView® Caspase-3 Substrates compared to other apoptosis assays is that it can be used to monitor capase-3 activity in real time.

Yes. We recommend a gentle fix in 2-4% paraformaldehyde for 10-15 minutes. Over-fixing can cause the signal to decrease. NucView® 488 staining can withstand permeabilization with 0.1% Triton X-100. Methanol fixation is not recommended.

Yes. NucView® Caspase-3 Substrates are compatible with flow cytometers and other instruments with the appropriate excitation and emission settings.

NucView® Caspase-3 Substrates have not been validated by Biotium for live tissue staining. However, we have had feedback from a customer who successfully used NucView® 488 Caspase-3 Substrate for live zebrafish embryo staining. NucView® Caspase-3 Substrates cannot be used in fixed cells or tissues.

As with all fluorescence based probes, bleaching will occur over time. How long you can view NucView® staining under the microscope depends on several factors including the initial signal strength and the intensity of the excitation source. Some users have reported monitoring NucView® for 4-5 days. You can see a time-lapse video of NucView® staining over 24 hours here:

https://biotium.com/technology/nucview-caspase-3-substrates/

The NucView® 488 Assay Kit for Live Cells (30029) contains NucView® 488 substrate at 0.2 mM in DMSO and caspase-3 inhibitor Ac-DEVD-CHO. The final DMSO concentration in the kit assay can be fairly high, which is undesirable for sensitive cell types. Because of this, we offer NucView® 488 Caspase-3 Substrate at 1 mM in DMSO (10402) and at 1 mM in PBS (10403), so customers can control the amount of DMSO in their assay. Note that in non-DMSO sensitive cell types, adding DMSO during the substrate incubation can enhance NucView® staining.

We also offer blue fluorogenic NucView® 405 in DMSO (10405) and PBS (10407), and orange fluorogenic NucView® 530 in DMSO (10406) and PBS (10408).

DEVD-CHO is a reversible inhibitor and may not sufficiently block caspase-3 activity. Adding an irreversible inhibitor like DEVD-FMK at the beginning of the experiment (before or at the time of apoptosis induction) may more effectively inhibit caspase activity.

We currently offer NucView® in three dye colors: green (our original NucView® 488), blue (NucView® 405), and orange (NucView® 530). Other colors of NucView® are in development.

Our NucView® substrates detect cleavage by caspases 3 and 7. We do not currently have NucView® substrates for other caspases.

Cationic dyes are widely used as mitochondrial probes. They accumulate within the cell and preferentially localize in the mitochondrial matrix, induced by the greater negative membrane potential of mitochondria in live cells compared to the plasma membrane potential. Membrane potential plays a direct role in governing the distribution of the dyes across the plasma membrane: the more negative the potential, the greater the accumulation of the positively charged dyes. MitoView™ 633 is a cationic lipophilic dye that is potential-dependent and accumulates in mitochondria in proportion to the electron gradient similar to classic dyes like TMRM, TMRE and Rhodamine-123.

Ratiometric dyes like JC-1 constitute another class of potential-dependent mitochondrial dyes. In normal healthy cells, JC‐1 accumulates in the mitochondria and undergoes aggregation in a potential‐dependent manner, whereas in unhealthy, apoptotic or dying cells with dissipated mitochondrial potential, the dye delocalizes to the cytosol where it dissociates to the monomeric state. The aggregated dyes (J-aggregates) fluoresce red, while the monomeric dyes fluoresce green.

Some mitochondrial‐specific dyes are considered “structural” dyes, as opposed to “functional” dyes, because they are capable of staining mitochondria regardless of their polarization status. The fluorescence of cells stained with these dyes is directly proportional to their mitochondrial content or “mitochondrial mass.” MitoView™ Green is a potential-independent dye that accumulates in the mitochondria and interacts with the mitochondrial matrix via hydrophobic interactions. It can be used to stain mitochondria in live as well as formaldehyde-fixed cells. MitoView™ Green staining is not compatible with solvent-based fixatives or permeabilization. Nonyl Acridine Orange (NAO) is another potential-independent mitochondrial dye that binds specifically to cardiolipin in the inner mitochondrial membrane. However, mitochondrial dye staining of fixed cells generally is less specific compared to live cell staining. For fixed cell staining, we recommend using one our CF® Dye conjugated mitochondrial marker antibodies.

The mechanism of binding for RedDot™ 1 and RedDot™ 2 to DNA has not been characterized. However, based on the dye structure, it may bind by a similar mechanism as DRAQ®5, which has been reported in the literature to be a concentration-dependent intercalator and minor groove binder.

DRAQ is a registered trademark of Biostatus, Ltd.

TrueBlack® and TrueBlack® Plus are hydrophobic in nature and quench lipofuscin autofluorescence mainly through hydrophobic interactions. They could therefore stain/bind to lipid structures and quench the fluorescence signal of BODIPY and other lipid droplet stains. Also, original TrueBlack® #23007 is used in 70% EtOH, which could interfere with lipid droplet morphology and affect staining. TrueBlack® Plus #23014, on the other hand, can be used in buffer instead of 70% ethanol, and may be more suitable for combining with lipid droplet staining. However, since the binding of TrueBlack® Plus is also dependent on hydrophobic interactions, we recommend testing TrueBlack® pre-treatment and post-treatment for compatibility with lipid droplet staining in your sample type.

Mitochondrial dyes, including MitoView™ Mitochondrial Dyes, are positively charged and lipophilic. They passively diffuse across cellular membranes and are presumed to accumulate in the mitochondrial matrix due to the proton gradient in the mitochondria (for a detailed review, see Cytometry Part A79A: 405-425, 2011).

However, some dyes are still retained in mitochondria after depolarization. Our dye chemists hypothesize that this is because some of the dyes are more lipophilic than others. Once they accumulate in the mitochondria because of their charge, they are less likely to diffuse back into the cytoplasm due to their hydrophobicity, even after the proton gradient that attracted them is dissipated by mitochondrial depolarization. Probably they associate with the mitochondrial membranes instead.

The so-called potential-independent dyes like MitoView™ Green, MitoTracker® Green, and Nonyl Acridine Orange are much more hydrophobic than potential-responsive dyes like MitoView™ 633, Rhodamine 123, and JC-1. The former dyes are retained after mitochondrial depolarization, and can be used to measure mitochondrial mass independent of potential. However, it would be more accurate to call these dyes relatively potential-insensitive, rather than potential-independent, because mitochondrial potential still plays a role in their localization. These dyes have been reported to show some loss of signal upon depolarization (Cytometry 39(3):203-10, 2000).

There is another class of mitochondrial dyes that accumulate in mitochondria based on charge, but also have a reactive group that can covalently link the dye to protein targets within the mitochondria, allowing them to be well-retained after fixation and permeabilization. Our MitoView™ Fix 640  is this type of dye.

Some dyes, like MitoView™ Green can stain mitochondria in cells that are already fixed. The mechanism by which this occurs is not well-understood. After fixation, there should be no proton gradient in the mitochondria to attract the dyes at all. Our chemists suspect that there may be some residual membrane potential in fixed mitochondria that is not due to the proton gradient (which would disappear following fixation), but instead arises from uneven distribution of proteins that have different isoelectric points (net charge). There are reports that the net charge of resident proteins in organelles differs based on the pH of the cellular compartment (Proc Natl Acad Sci USA 115(46):11778-11783, 2018). Charge differences may be sufficient to attract cationic lipophilic dyes to mitochondria in the absence of a proton gradient, due to a combination of weak electrostatic and hydrophobic interactions with mitochondrial proteins and membranes.

However, currently there is no direct evidence to suggest this is the mechanism for MitoView™ Green staining of fixed cells. There may be other targets that the dye is binding. For example, Nonyl Acridine Orange is reported to bind cardiolipin, a lipid that is enriched in mitochondrial membranes. It’s possible that MitoView™ Green binds to particular molecules in mitochondria with some degree of specificity. However, staining of fixed cells with mitochondrial dyes generally is not as specific as staining of live cells. That’s why we recommend using mitochondrial marker antibodies instead of dyes to stain fixed cells when possible.

Most of our MitoView™ dyes are sensitive to mitochondrial membrane potential to some degree, and therefore are recommended for use in live cells only.

Our MitoView™ Fix 640 dye is designed to be fixable after staining of live cells. Staining is compatible with formaldehyde fixation, detergent permeabilization, methanol fixation, and subsequent immunofluorescence staining. MitoView™ Fix is not recommended for staining cells that are already fixed, because staining is non-specific in fixed cells.

MitoView™ Green is relatively insensitive to mitochondrial membrane potential, and can be used to stain formaldehyde-fixed cells (for best results, we recommend first fixing, then staining). MitoView™ Green does not stain well with samples that are fixed with methanol, permeabilized with detergent, or paraffin embedded. Nonyl Acridine Orange also stains mitochondria in fixed cells, but the staining of fixed cells is not specific to mitochondria (cytoplasmic and nuclear staining is also observed). In general, staining of fixed cells with mitochondrial dyes is less specific than live cell staining. For best results when staining mitochondria in fixed cells or tissue sections, we recommend using one of our Mitochondrial Marker Antibodies, available with a wide selection of bright and photostable CF® Dyes and other conjugations.

LysoView™ dyes are fluorogenic dyes that contain weakly basic amines and accumulate in the low pH environment of lysosomes as well as other acidic compartments in the cell including early and late endosomes. Although the fluorescence of LysoView™ 540 and LysoView™ 640 is sensitive to increasing pH, it would be hard to specifically distinguish early from late endosomes or lysosomes based on florescence intensity. Using antibodies against early or late endosomal markers or compartment-specific  (GFP or other) fusion constructs would be more appropriate.

Other options that may be suitable for monitoring endocytosis and vesicle trafficking include the CF® Dye Dextrans, CF® Dye Hydrazides, CF® Dye Human Transferrin Conjugates, CF® Dye Cholera Toxin Subunit B, and Nerve Terminal Staining Kits.

ViaFluor® Live Cell Microtubule Stains are cell-permeable taxol probes for imaging the microtubule cytoskeleton in live mammalian cells. We do not have permeability data for these probes to the plant cell wall. Based on literature, plant tubulin binds taxol weakly as compared to animal tubulin, so staining may be weak or non-existent. We do offer low-cost trial sizes for evaluation.

Intracellular lipid droplets are cytoplasmic organelles involved in the storage and regulation of lipids. The LipidSpot™ Lipid Droplet Stains are fluorogenic neutral lipid stains that rapidly accumulate in lipid droplets. They become brightly fluorescent in the presence of neutral lipids like triglycerides and cholesterol esters.

LipidSpot™ stains may be used on fresh or PFA-fixed tissue cryosections, similar to Nile Red or BODIPY dyes. However, lipid droplets may not be well fixed in tissue sections, so discrete lipid droplet staining may not be preserved. Paraffin embedded sections are not recommended, because paraffin tissue processing extracts cellular lipids. Similarly, methanol or acetone fixation of cryosections may extract lipids, and is not recommended.

Mounting medium also may disrupt LipidSpot™ staining. We’d recommend either mounting tissue sections in PBS, or using a glycerol-based mounting medium like EverBrite™ Mounting Medium, and imaging within one day of mounting. FluoroShield Mounting Medium is not compatible with LipidSpot™ staining.

Mounting medium can alter the staining of lipophilic dyes like LipidSpot™, due to interaction of the dyes with glycerol or other components that help form the interface between the coverslip and slide. The antifade compounds in mounting medium are generally compatible with the dyes. In our tests, LipidSpot™ staining was well preserved in EverBrite™ Mounting Medium (catalog. nos. 23001/23002) for up to 24 hours after mounting, but lipid droplet size and staining intensity were somewhat altered after samples were stored in mounting medium for several days. Therefore, if mounting medium is required to image samples, we’d recommend imaging as soon as possible after mounting.

LipidSpot™ is not compatible with FluoroShield mounting medium (staining is lost immediately after mounting). We have not tested other types of mounting medium.

PMA is stable after dilution to 0.2 mM in water as long as it is protected from light and can be stored in the same way as the 20 mM stock solution.

Note: Do not remove the cover or introduce liquids to the interior of the PMA-Lite.

1. Thoroughly wipe all exposed PMA-Lite surfaces and the inner rims of the tube holes with 10% bleach in water (household bleach diluted at a ratio of 1 part bleach to 9 parts water).
2. Let the bleach sit on the unit for 10 minutes.
3. Thoroughly wipe the surfaces with dH2O.
4. Wipe the surfaces with 70% ethanol and allow to air dry.

Viability PCR originally used EMA (ethidium monoazide) to inactivate dead cell DNA. Biotium developed PMA (propidium monoazide) in collaboration with investigators at Montana State University (Nocker et al. 2006). PMA is more selective for dead cells than EMA, and became widely used for selective detection of viable microbes and viruses. PMAxx™ is Biotium’s newest viability PCR dye, designed to be more effective than PMA at eliminating PCR amplification of dead cell DNA. Therefore it provides the best discrimination between live and dead bacteria.

Learn more about PMA and PMAxx™ for viability PCR.

Both PMA and PMAxx™ are both offered as 20 mM solutions in water. We also offer PMA as a solid, and customers often ask whether it needs to be dissolved in DMSO. While in some publications users have prepared their PMA stock solution in DMSO, this is not necessary. Unlike the older viability dye EMA, which is not soluble in water, PMA and PMAxx™ are very water soluble.

We have compared PMA and PMAxx™ for their ability to differentiate between live and dead cells in viability PCR assays, and in all bacteria strains tested*, PMAxx™ had better activity. Therefore, while we haven’t tested every bacterial strain, we recommend PMAxx™ for bacterial viability PCR.

A recent publication by Randazzo et al. compared several viability PCR dyes in norovirus, and found that PMAxx™ worked the best for that organism.

We have also compared PMA and PMAxx™ in viability PCR assays with the yeast Saccharomyces cerevisiae, and found that PMA was equal to or better than PMAxx™. Therefore we recommend PMA for yeast and fungus viability PCR.

*E. coli, Salmonella enterica, Listeria monocytogenes, Bacillis subtilis, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Lactobacillus casei.

Many users have had success using PMA in viability PCR with environmental samples, but these samples can be more challenging than pure cultures. We’ve outlined some of the challenges below.

Water samples In order to perform viability PCR on water samples, some users have developed methods to concentrate the microorganisms onto a 0.45 um filter, and then treat the filter with PMA. An example protocol can be found in this publication: Ditommasso S., et al. (2015) Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths. Mol. Cell Probes. doi: 10.1016/j.mcp.2015.05.011. 

Opaque or complex samples Viability PCR with PMA has been successfully reported in various complex sample types, such as sewage or soil. No reports have been made yet with PMAxx™ in complex samples, but we would expect it to work as well or better than PMA. The main considerations for complex samples are dye concentration and light penetration. Typically a higher dye concentration (100 uM or more) is needed because some dye may bind to contaminants in the sample. Longer light treatment and more mixing during light treatment may be required, since it will be harder for the light to penetrate through the sample. An example protocol can be found in this publication: Guo F. and Zhang T. (2014) Detecting the nonviable and heat-tolerant bacteria in activated sludge by minimizing DNA from dead cells. Microb. Ecol. dio 10.1007/s00248-014-0389-2.

Viability PCR using PMA has been published in hundreds of publications, with dozens of different organisms, and many different types of samples. For more information, please see our PMA publication list or do a literature search for your application of interest.

PMA and PMAxx™ dyes are chemically stable as long as they are protected from light (stored in the original amber vial, for example). We have confirmed by HPLC analysis that the dyes are unaffected after being left for 10 days at ambient temperature.

In our standard viability PCR assay protocols we say that following PMA treatment, DNA should be purified from the cells before being used as a template for PCR. However there have been reports of the successful use of bacterial cell lysates in viability PCR following PMA treatment. The main consideration is to ensure that all of the dye is efficiently photolysed before preparing the lysate.  Unreacted PMA dye in the lysate could potentially inhibit the PCR reaction by binding to the DNA or polymerase. If you are considering trying this, we would suggest pelleting the cells after dye and light treatment to remove the excess free dye from the solution. In addition, control experiments should be performed to determine the ideal PMA and light treatment conditions that result in no PCR inhibition.

PMA and PMAxx™ are membrane-impermeant dyes that selectively modify DNA in dead cells with damaged membranes. Therefore, samples should not be frozen before PMA treatment, because this will permeabilize live cell membranes to the dye, and live/dead discrimination will be lost.

After PMA treatment and photolysis, PMA or PMAxx™ becomes covalently linked to dead cell DNA, and any excess dye in solution should be photolyzed and rendered non-reactive. Cells can be frozen at this point for storage before DNA extraction and PCR. For full cell recovery, we would recommend pelleting the cells and removing the supernatant before freezing.

PMA and PMAxx™ dyes are very sensitive to light. Light exposure, even ambient room light, causes a chemical change in the dye molecule. Therefore we recommend that vials of PMA or PMAxx™ dyes only be opened in darkened rooms or very dim light. We always suggest that you perform a small test experiment if you are concerned that a dye may have lost activity.

For some strains of bacteria, we sell complete viability PCR kits, which include strain-specific primer sets that have been validated at Biotium, as well as in publications in most cases. However any validated primer sets may be used to target your species of interest. We and others have found that amplicon length can affect your viability PCR results. Using longer amplicons generally results in better suppression of dead cell PCR products. For an example see this publication: Banihashimi A., et al. (2012) Long-amplicon propidium monoazide-PCR enumeration assay to detect viable Campylobacter and Salmonella. J. Appl. Miobiol. dio 10.1111/j.1365-2672.2012.05382.x.

While you can perform viability PCR with amplicons as short at 100 bp, we advise that you use longer amplicons for better results.

No, the PMA Enhancer should only be used when only gram- bacteria will be studied. The PMA Enhancer has a detrimental effect of gram+ bacteria.

Biotium offers the PMA-Lite™ LED Photolysis Device for light-induced cross-linking of PMAxx™ and PMA to dsDNA. The PMA-Lite™ is designed for samples in microcentrifuge tubes.

Commercial halogen lamps (>600 W) for home use have been employed for photoactivating PMA in some publications, though results may be less consistent due to variation in the set-up configurations. If you decide to use a halogen lamp, place the tubes on a block of ice on a rocking platform to ensure continuous mixing. We recommend freezing water in a clear tray to make the ice block, and placing a piece of aluminum foil under the tray to reflect light upward toward the bottom of the tubes. Set the lamp 20 cm above the samples, so that the light source is pointing downward onto the samples (up to 45° downward slant is OK). Expose samples to light for 5-15 min while rocking.

The LEDs in the PMA-Lite™ and PMA-Lite™ 2.0 have a wavelength that is 465-475 nm and a brightness of approximately 600-800 millicandela (mcd). These are nominal values provided for reference use only, individual LED wavelength and brightness are not a calibrated specifications for the device.

There are three LEDs in each well (one bottom, two side) that provide illumination around each sample tube for efficient photoactivation.

The illumination in each well on the PMA-Lite  far exceeds what is required for photocrosslinking of the viability dyes EMA, PMA, or PMAxx™ to nucleic acids. Therefore, any variability in brightness of the PMA-Lite LEDs should not significantly affect the v-PCR results. If performance verification is required, we recommend doing a functional PMA-PCR assay to verify that PMA-treated samples photoactivated in the device give qPCR results within an acceptable range. Mixing the samples during photoactivation and using longer illumination times may be necessary if the samples are complex and not fully transparent to light.

For other related FAQs, see Is illumination even across all positions in the PMA-Lite™ device? and Can I use PMA or PMAxx™ with environmental samples?

Each tube position on the PMA-Lite™ is illuminated by the three LED bulbs. We haven’t tested positional variability, but it is likely that the illumination varies slightly between positions and between devices. However, the illumination at each position is exceedingly bright, far in excess to what is required for photocrosslinking of the viability dyes EMA, PMA or PMAxx™ to nucleic acids, so any variability should not significantly affect the vPCR results.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF® Dye succinimidyl ester stability

Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Some products are shipped with blue ice packs as an extra precaution against high temperatures. The blue ice packs may be thawed upon arrival without affecting product performance.

Products with recommended storage at ultra low temperature (-70°C) that also ship on dry ice should arrive frozen. If a product you received was shipped on dry ice and thawed during transit, please contact customer service at order@biotium.com.

You can also download our Product Storage Statement here.

For dyes or reagents that are supplied lyophilized (as solids), it is hard to compare quantities based on appearance of the dye in the tube, because during the lyophilization process the dye can dry down in different ways, either spread out all over the tube, clumped together, or coating the sides or bottom of the tube. Centrifugation of the tube may not help in collecting the dye solid to the bottom of the tube as this generally works for solutions. However, lyophilized solids are packaged based on highly accurate absorbance measurement of the reagent solution prior to drying, so the vial will contain the correct amount of dye.

Coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

Biotium ships all antibodies (primary, secondary and conjugates) at room temperature. We guarantee their quality and performance under these conditions based upon our stability testing. Antibodies were subjected to accelerated stability testing by storing them at various temperatures (4°C, room temperature, or 37°C) for 1 week to mimic simulated shipping conditions and tested in immunostaining experiments. All antibodies showed the expected brightness and specificity, even after storage at sub-optimal temperatures for a week or longer. You can also download our Product Storage Statement here.

In line with our goal to be more environmentally friendly by reducing the use of excess packaging, and lowering shipping costs for our customers, products that have passed our stability testing are shipped at room temperature.

Once you have received the antibody vial, please follow the long-term storage instructions on the product information (PI) sheet.

The storage conditions that a Biotium label recommends are deliberately conservative. Long term storage at low temperatures usually will increase the time that the product will remain within specification, unless specific instructions otherwise are provided (such as “do not refrigerate” or “do not freeze”).

Biotium labels and product information sheets may show a specific temperature for long term storage, or a temperature range that is adequate for long term storage. For all products, the implied range is as follows for the following storage conditions:

• Room Temperature
  For Biotium products where the label indicates room temperature or RT, this implies storage in ambient conditions between 20°C and 30°C.

• Refrigerator
  For Biotium products where the label indicates 4°C, this implies storage in a refrigerator that is between 2°C and 8°C.

• Freezer
  For Biotium products where the label indicates -20°C, this implies storage in a freezer that is between -35°C and -5°C.

• Ultra Low Temperature Freezer
  For Biotium products where the label indicates -70°C, this implies storage in an ultra low-temperature (ULT) freezer that is below -60°C.

Some of our large volume light-sensitive products are packaged in amber bottles or amber glass vials, which are easy to handle and protect the products from light. However, amber micro packaging vials (0.5 mL or 2 mL) make it very difficult to see small quantities of dye when preparing solutions or pipetting. Therefore, we only package photoreactive dyes like PMA or PMAxx™ in amber micro packaging vials. Some of our reactive dyes are packaged in transparent vials, then sealed inside a moisture-resistant foil bag, which also will protect them from light.

Other fluorescent dyes in transparent vials should stored in the dark for long term storage. It’s fine to store dye vials uncovered in a windowless refrigerator or freezer with an automatic light shut-off. If dyes will be stored in a glass-front (deli-style) refrigerator, walk-in refrigerator/freezer, or at room temperature with constant light exposure, they should stored in a non-transparent box (like a white cardboard freezer box), in a closed drawer, in a black plastic bag, or covered with aluminum foil.

Fluorescent dyes generally are not sensitive to brief light exposure while they are being handled on the bench during an experiment. To be on the safe side, we usually loosely cover tube racks with a piece of foil if the dye vials are going to be out on the bench for more than 30 minutes or so. But most dyes are stable enough that even accidentally leaving them on the bench for a day will not affect performance. When handling photoreactive dyes like EMA, PMA, or PMAxx, we take the extra precaution of dimming the lights.

Compound solubility is listed on product web pages, product information sheets or product safety and data sheets. Check the product information sheet for specific instructions for preparing stock solutions. Stock solutions are generally prepared at 10X-1000X the final required working concentration, typically in the range of 1-100 mg/mL. For dyes and indicators, we generally prepare a concentrated stock solution at 5-10 mM in the recommended solvent. The dye can then be diluted to the final desired concentration in the buffer or medium used for the application.

Solubility definitions

To dissolve lyophilized compounds, simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.

Difficult to dissolve compounds can be heated to 50°C or higher (note: we do not recommend heating coelenterazine solutions), and vortexed or sonicated until completely dissolved. Some chemical compounds are just kinetically slow to dissolve. This can be especially true when the compounds are highly pure and in crystalline form. An alternative and gentler way to dissolve these compounds can be to simply rock the compound/solvent mixture in the dark overnight.

When preparing stock solutions in organic solvents such as DMF, DMSO, or alcohol for use in living cells or organisms, make a concentrated stock so that the final concentration of solvent in the working solution will not be toxic. A general guideline for immortalized cell lines is to keep the final solvent concentration below 1%; certain cell lines, primary cells or experimental organisms may be more sensitive to solvent toxicity. Similarly, for enzymatic reactions, the final concentration of solvent should be kept below 1% to avoid inhibiting enzyme activity.

Compounds dissolved in aqueous solution can be sterilized by filtration if necessary. Generally, concentrated stock solutions of compounds in organic solvents do not require sterilization, but culture medium can be sterilized by filtration after addition of the compound.

Some of our products are packaged from a solution followed by solvent evaporation or lyophilization. If the chemical compound is very lightly colored or colorless and in small quantity, it may become thinly coated on the wall of the vial, making the vial appear empty. So, before you ask for a replacement, please inspect the vial carefully.

To dissolve lyophilized compounds, simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.

Note that blue fluorescent dyes such as CF™350, CF™405M, and CF™405S are colorless or very pale yellow, and may be difficult to see.

Many of our solid compounds are packaged by lyophilization, in which case they usually do not appear as fluffy powders, but form a film or coating on the sides of the vial. Simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.

The AccuOrange™ assay is a fluorescent dye-based assay. The dye binds to proteins primarily through hydrophobic interactions. Proteins denature upon heating; the dye binds to the exposed hydrophobic pockets of the protein after cooling. The free AccuOrange™ dye is fluorogenic due to non-radioactive decay but becomes highly fluorescent due to the rigid conformation inside the pocket.

The AccuOrange™ assay more sensitive than traditional protein quantitation assays such as BCA, Bradford and Lowry, and shows superior linearity and reproducibility than the NanoOrange® protein quantitation assay (Thermo Fisher Sci.), but has low tolerance for detergents like SDS and Triton® X-100.

Even though AccuOrange™ buffer does contain SDS, which is required for the dye to bind proteins, the assay is very sensitive to small changes in SDS concentration, and also cannot tolerate non-ionic detergents that form mixed micelles with SDS, like Triton®. Therefore we don’t recommend using the kit for cell lysates or other samples with significant amounts of detergents.

Use of pre-stained protein markers can quench fluorescent protein stains. Switching to an unstained marker should resolve the issue.

We do not recommend staining proteins on the polyacrylamide gel with the One-Step protein gel stains before western blotting as it can significantly reduce protein transfer to the membrane. If protein detection on the gel prior to transfer is desired, the Total Protein Prestains would be more suitable.

The VersaBlot™ Total Protein Normalization Kits allow simple, sensitive and highly linear protein quantitation on SDS-PAGE gels and western blot membranes. The kits allow you to label purified proteins or cell lysates with our near-infrared CF® dyes before running the samples on SDS-PAGE. After electrophoresis, the bands can be visualized on the gel using a fluorescent gel scanner, allowing detection of as little as 1 ng protein per band. Labeled proteins also can be transferred to membranes for western blotting. The staining demonstrates excellent linearity for quantitation of total protein over a wide dynamic range, outperforming traditional western blot normalization based on housekeeping protein detection and staining is reversible.

Lumitein™ is a luminescent dye designed for detecting proteins in SDS polyacrylamide (SDS-PAGE) gels. It can also be used to detect proteins in native PAGE gels after an additional SDS incubation step as described in the protocol PI-21002.

Gels stained with One-Step Blue® can be dried just like gels stained with Coomassie. The stain will not interfere with the detection of radiolabeled proteins.

MTT and XTT are colorimetric based assays, while resazurin can be measured using colorimetric or fluorescence detection. MTT is not a soluble product, so the cells must be lysed to solubilize the formazan salt before absorbance can be measured. XTT and resazurin do not require cell lysis, allowing kinetic monitoring of the same samples at different timepoints.

AlamarBlue® contains resazurin and additional compounds to prevent the over-reduction of resazurin to a non-fluorescent product. These additives also slow the rate of generation of the fluorescent product. Consequently, the alamarBlue® assay requires longer incubation times compared to resazurin.

Resazurin is reduced by cells to the fluorescent product resorufin. Resorufin can be reduced further to a non-fluorescent compound. Therefore the densest wells may have lowest fluorescence due to over-reduction of the substrate. Please see the product information sheet for more details. The kit protocol provides general guidelines and may need to be optimized empirically for your experimental system. You may need to vary cell density or assay incubation time to ensure that your samples fall in the linear range of the kit.

Medium with phenol red is compatible with the resazurin assay. Phenol red does not interfere with the resazurin reaction nor does it affect detection.

Contamination from genomic DNA is a big concern because it can interfere with downstream applications. The RNAstorm™ kit includes an optimized DNase digestion step that removes contaminating genomic DNA without significantly affecting RNA yield. While this step is optional, it is highly recommended.

The most significant variable that affects the total amount of RNA or DNA obtained is the quality of the sample itself (i.e., the type and amount of tissue and the care taken in isolation and preservation of the sample). Assuming a reasonable sample quality, you can expect to obtain more than 1 ug using the RNAstorm™ or DNAstorm™ kits.

Yes. Good quality libraries can be obtained, providing that the RNA is of sufficiently high quality. For Illumina sequencing, a DV200 of at least 30% is recommended, and samples that provide at least 1 µg of RNA should be used.

Use a microtome to obtain 5-10 µm sections from FFPE samples. Sections thinner than 5 µm may be used if they can be reliably cut. Sections thicker than 10 µm are not recommended because they may not be fully digested. Also, no more than 5 sections (10 µm each) should be used for each extraction. Using too much tissue can lead to incomplete digestion and reduced yields.

Yes, tissue that is not embedded in paraffin can be used with these kits. In this case, we recommend mechanically grinding an amount of tissue equivalent to the recommended number of sections. If you are isolating RNA, we also have an RNAstorm™ kit for isolating RNA from fresh or frozen cells or tissue samples.

The RNAstorm™ FFPE and DNAstorm™ FFPE extraction kits include a recommended Deparaffinization Reagent. Unlike other common methods (e.g., xylenes), the Deparaffinization Reagent is efficient, non-toxic, and does not require the use of a fume hood. In our testing, the included reagent is at least as effective as xylenes at removing paraffin and allowing purification of high-quality nucleic acids.

FFPE-derived RNA is much more challenging to quantitate accurately than RNA obtained from fresh samples. It is not enough to know the absolute amount of RNA that is present, but also whether the RNA will work in downstream applications, which depends on the following factors:

• Fragment size distribution: a 5 µg sample (as measured by Qubit) can be useless for RNA-Seq if it consists of fragments < 200 nt.
• Chemical modification: for RNA obtained from formalin-fixed samples, various chemical adducts and crosslinks, including base modifications, base-base crosslinks, and base-protein crosslinks can make nucleic acid molecules inaccessible to enzymes and therefore inactive in downstream applications.
• Contamination: cellular debris, proteins, salts, and detergents used during purification can bias downstream assays. For example, UV/Vis-based methods such as Nanodrop are particularly susceptible to contaminants that absorb in the 200-280 nm range.
• Fluorescence-based methods such as Qubit are liable to significant error. When working with low concentrations of DNA or RNA, dye-based detection may not be linear. One must also be mindful of contamination by genomic DNA in an RNA sample because the dyes used for fluorescence quantitation are not entirely specific for FFPE-derived DNA or RNA.
• Quantitative PCR is the preferred method for quantitation of heavily damaged and modified nucleic acids.

Due to the wide size distribution of DNA isolated from FFPE tissue samples, we recommend using pulsed-field gel electrophoresis (PFGE). Methods based on capillary electrophoresis such as the Agilent Bioanalyzer can also be used but may not properly resolve high molecular weight fragments (greater than 10kb) in better-quality samples.

Yes. Good quality libraries can be obtained, providing that the DNA is sufficiently high quality.

Contamination from RNA is eliminated by performing an optimized RNase digestion step immediately following the lysis step.

Although the RIN number can provide general information about the extent of sample fragmentation, it is not sensitive or predictable enough to be a useful indicator of downstream performance, especially for RNA-Seq. Very often, RIN numbers for FFPE-derived RNA will be between 2 and 3. Some of these samples will be useful for RNA-Seq, and others won’t – the RIN will not tell you, however.

A slightly better predictor of performance in RNA-Seq using Illumina sequencing is the DV200, which represents the percentage of RNA fragments longer than 200 nucleotides. The DV200 is also calculated based on Bioanalyzer data, but suffers from the same drawbacks as all Bioanalyzer-based methods, specifically high variability.

• Avoid methods based on organic solvents (Trizol)
• Avoid harsh chaotropic salts (i.e. guanidinium)
• Avoid detergents that impact downstream quantitation by UV and/or Qubit (e.g. Triton X-100)
• Do not rely on RIN to quantitate the integrity of an FFPE-derived sample, use DV200 instead.
• Use a kit or method that removes chemical modifications from formalin. Do not raise the temperature to 80˚C or above. Even short times at this temperature will significantly lower integrity.
• Be wary of Qubit and Nanodrop concentrations because of the possibility of contamination by organic molecules or DNA.
• Use qPCR to quantitate your RNA, and always look carefully at melt curves to determine whether nonspecific amplification may have occurred.

PCR inhibition is often observed when high amounts of FFPE-extracted template DNA are used. The inhibition is not usually due to the presence of contaminants but results from residual chemical modifications and damage in the DNA itself. Several simple adjustments to the PCR protocol can overcome this issue. First, the amount of template DNA should be decreased. Second, the amount of PCR polymerase should be increased by 2-4X. Third, the annealing and extension times should be extended. Fourth, the amount of dNTPs can be increased.

An in-depth discussion of this issue is found in Dietrich et al. (2013), PLoS ONE 8(10): e77771.

Yes, the RNAStorm™ and DNAStorm™ FFPE kits may be used sequentially. The steps below will allow the protocol to be adapted to extract both RNA and DNA from one sample. You can also download this information in an app note.

Begin by extracting the sample according to the RNAstorm™ kit protocol with the following modifications:

1.  Perform step 3 (normally a 2 hour incubation) for only 30 minutes at 72˚C. See note below regarding possible optimization of this step.
2.  Perform steps 4 and 5 of the RNAstorm™ protocol as directed, but do not discard the pellet (which contains the DNA) in step 5.
3.  Transfer the supernatant (which contains the RNA) to a new tube as instructed in step 6.
4.  Continue to incubate the supernatant for another 1.5 hours at 72˚C (2 hours total including the initial 30 minutes), then proceed with step 7 of the RNAstorm™ protocol (add Binding Buffer) and all remaining steps as instructed.
5.  Use the pellet from step 2, which contains DNA, as input for step A5 (or B8, depending on deparaffinization choice) of the DNAstorm™ kit manual.
6.  Continue with step A5 (or B8) of the DNAstorm™ protocol by adding 200 µL of CAT5™ Buffer to the pellet, then continue as instructed by the DNAstorm™ protocol.

Note: the initial incubation period can be adjusted depending on relative DNA and RNA yields. If the RNA yield is high but the DNA yield is low, reduce the incubation time in step 3 (no less than 15 mins). If the DNA yield is good but the RNA yield is low, increase the incubation time in step 3 (no more than 2 hours).

The maximum capacity of the spin columns in the kit is similar to a standard miniprep column, about 20 ug of DNA. However, the expected yield from FFPE extraction is much lower, it is very rare to get more than 1-2 ug of DNA per prep.

The CELLDATA RNAstorm™ 2.0 FFPE RNA Extraction Kit (Cat. No. CD506) and CELLDATA DNAstorm™ 2.0 FFPE DNA Extraction Kit (Cat. No. CD507) contain a Dewaxing Solution that was reformulated for IP considerations. These kits are direct replacements for the discontinued CELLDATA RNAstorm™ or DNAstorm™ FFPE Extraction Kits (CD501, CD502). The kits continue to utilize Biotium’s proprietary CAT5™ catalytic technology for efficient reversal of formaldehyde crosslinks.

Unless specified as isotype-specific, anti-IgG secondary antibodies are raised against whole immunoglobulin, so they are expected to cross-react widely with isotypes other than IgG.

No, you don’t always need to use serum from the same species as the secondary antibody for blocking. BSA, fish gelatin, goat serum, or non-fat milk (for western) can be used with secondary antibodies from most host species. However, it is important to avoid using blocking serum from the same host species as your primary antibody that is detected by a secondary antibody against the same species (immunoglobulins in the blocking serum will compete for the secondary binding). For example, avoid using goat serum for blocking if you are using a goat primary antibody with anti-goat secondary antibody.

Our TrueBlack® IF Background Suppressor System and TrueBlack® WB Blocking Buffer Kit contain proteins from non-mammalian sources, and should not interact with secondary antibodies from mammalian or chicken host. Our BSA also is immunoglobulin-free and should not interfere with secondary antibodies.

H+L stands for heavy and light chains. It means that the antibody will bind to epitopes on both the heavy chains (constant regions) and light chains (variable regions) of the target species immunoglobulin.

“Min x” followed by a list of species means that the antibody has minimal cross-reactivity against antibodies from those species. The antibody has been adsorbed against serum proteins from the listed species, to ensure that it does not cross-react with those species.

Highly cross-adsorbed antibodies are recommended when doing multiple staining with antibodies from different species (for example, co-staining with two different primary antibodies from mouse and rat hosts) or when staining tissues from species that are closely related to the antibody host (such as using mouse antibodies to stain rat tissue).

We recommend using highly cross-adsorbed secondary antibodies whenever you perform multiple staining with two primary antibodies from different hosts. It is particularly important to use highly cross-adsorbed antibodies when staining with antibodies from two closely related species, such as mouse and rat.

Using highly cross-adsorbed secondary antibodies when staining tissues from a species with primary antibodies from a closely related species (for example, staining rat tissue with mouse primary antibody), will prevent background from cross-reactivity of the secondary with endogenous immunoglobulin in the tissue. This is important when staining immune tissues, but background from endogenous immunoglobulin is common in other tissues around blood vessels, where immunoglobulin also is abundant.

As a rule, we recommend using secondary antibodies from the same host for multiplex staining (for example, combining Goat Anti-Rabbit with Goat Anti-Mouse, or Donkey Anti-Rabbit with Donkey Anti-Mouse). If we don’t currently offer the particular secondary antibody conjugate you wish to use, be sure to contact us, we may be able to perform a custom conjugation for you.

In our experience, cross-reactivity between donkey and goat host secondary antibodies is low, but this is not guaranteed to be the case for every lot of antibody. If you decide to combine secondary antibodies from different hosts, we recommend performing the appropriate controls to make sure the two secondary antibodies are not cross-reacting with each other.

No, antibodies designated as anti-IgG (H+L) with no subtype specified will cross react with all sub-types of IgG, as well as other isotypes like IgM, because they react with epitopes on the both the heavy (constant) and light (variable) regions of the antibody.

Secondary antibodies that react with IgG (H+L) will react with epitopes on both heavy and light chains, so they will react with other isotypes of primary antibody, such as IgM, or different subtypes of IgG (IgG2, IgG2a, etc.). Secondary antibodies that specify a specific isotype for their reactivity (such as Goat Anti-Mouse IgG2a, Isotype Specific) are cross-adsorbed against other isotypes for specific binding.

Isotype-specific antibodies (for example, Goat Anti-Mouse IgG1, Goat Anti-Mouse IgG2a, or Goat Anti-Mouse IgG2b) can be used to specifically detect primary antibodies from the listed sub-class. This can be useful for multiplex detection using multiple mouse monoclonal antibodies that are different isotypes.

Anti-mouse subtype-specific secondary antibodies also can be useful for avoiding background when staining mouse tissues with mouse monoclonal antibodies, because IgG is present at relatively low levels in most mouse tissues.

Isotype-specific antibodies also can be used for identifying the isotype of a monoclonal antibody, or for staining B-cell subsets.

IgY is the avian counterpart to mammalian IgG, or the type of antibody produced in birds. Goat Anti-Chicken IgY (H+L) is a secondary antibody raised in a goat host that reacts with both heavy and light chains of chicken antibodies.

F(ab) fragment-specific antibodies react with the light chains and not the heavy chains of immunoglobulins from the target species. They can be used to detect antibody fragments, or to avoid cross-reactivity with immunoglobulin heavy chains.

F(ab’)2 is a fragment of IgG that is prepared by pepsin digestion of IgG. F(ab’)2 fragment is the disulfide-linked heterodimer of the two light chain dimers, so it retains bivalent epitope binding like whole IgG, but because it lacks the heavy chains, it is smaller in size (~110 kDa compare to 150 kDa for whole IgG).

F(ab’) is a monovalent fragment consisting of a single light chain homodimer, which is obtained by pepsin digestion of IgG, followed by reduction of the light chain disulfide bond.

F(ab’)2 and F(ab’) fragments do not bind to immunoglobulin receptors on cells, which can be useful for achieving specific staining of the primary antibody target. The fragments also will not bind Protein A or Protein G.

TrueBlack® Lipofuscin Quencher can be used with the TrueBlack® IF Background Suppressor (Permeabilizing) kit in an immunofluorescence staining workflow, to reduce background from both non-specific antibody and dye binding, and autofluorescence arising from the sample itself. Because TrueBlack® IF reagents contain detergent, the TrueBlack® lipofuscin quencher would need to be used after immunostaining (Post-treatment protocol) as the latter is not compatible with detergents present in the IF reagents.

If immunostaining is performed with buffers lacking detergents, the TrueBlack lipofuscin quencher may be used before (Pre-treatment protocol) or after immunostaining.

TrueBlack® Lipofuscin Autofluorescence Quencher and TrueBlack® Plus Lipofuscin Autofluorescence Quencher were not designed for colorimetric detection, but they will stain lipofuscin black and be visible in light microscopy.

The mechanism of action of TrueBlack® and TrueBlack® Plus Lipofuscin Autofluorescence Quenchers is similar to the traditionally used dye Sudan Black B. Lipofuscin consists of autofluorescent granules of oxidized proteins and lipids that build up in the lysosomes of cells as a consequence of aging. The TrueBlack® lipofuscin quenchers are hydrophobic in nature and associate with the lipid rich lipofuscin to quench autofluorescence.

TrueBlack® and TrueBlack® Plus Lipofuscin Quenchers have similar properties as Sudan Black B and, therefore, primarily stain lipofuscin. Like Sudan Black B, they may also stain some myeloid cells, as well as lipid droplets, but we have not confirmed this.

TrueBlack® Plus Lipofuscin Autofluorescence Quencher is the only lipofuscin quencher that can be used in aqueous buffer instead of 70% EtOH, allowing longer incubation times for thick tissue samples without shrinkage. TrueBlack® Plus offers even lower far-red background than the original TrueBlack® Lipofuscin Autofluorescence Quencher. It is recommended to review the staining protocol of TrueBlack® and TrueBlack® Plus to decide between the two.

While customers have reported success using TrueBlack® Lipofuscin Autofluorescence Quencher to quench RBC autofluorescence, during in-house research we’ve only ever seen partial quenching for RBCs. Experimental samples can vary quite a lot in the intensity of autofluorescence depending on age and processing, so the effectiveness of RBC quenching may be variable.

When we test TrueBlack® Lipofuscin Autofluorescence Quencher in an assay, we evaluate lipofuscin quenching in human brain sections, but our standard QC for the dye is measuring purity and OD of the solution, not tissue staining.

Alternative products that may be more effective at quenching RBCs or ECM would be TrueBlack® Plus (Cat. No. 23014) or EverBrite™ Hardset with TrueBlack® (Cat. No. 23017-23019) however, these products have not been empirically evaluated for this application.

TrueBlack® Lipofuscin Autofluorescence Quencher (Cat. No. 23007) and TrueBlack® Plus Lipofuscin Autofluorescence Quencher (Cat. No. 23014) are designed to reduce autofluorescence from lipofuscin in tissue samples such as mouse and human brains and retina. While TrueBlack® Lipofuscin Quenchers have been reported to reduce autofluorescence from other sources, such as collagen, elastin, red blood cells, and general background fluorescence, they are not as effective at quenching these types of autofluorescence as for lipofuscin autofluorescence. However, they may improve background from a variety of sources in different experimental systems.

TrueBlack® IF Background Suppressor System (Cat. No. 230120) is a buffer system designed for optimal blocking of non-specific antibody binding as well as direct interaction of fluorescent dyes on antibodies with cells or tissue sections to eliminate non-specific staining for immunofluorescence (IF).

The TrueBlack® WB Blocking Buffer Kit (Cat. No. 23013) is a ready-to-use buffer system for blocking non-specific interactions of dye-labeled antibodies with proteins and the blotting membrane in fluorescence-based western blotting (WB).

Dyes that carry multiple negative charges can introduce background. Usually, this is more of a concern with labeled antibodies that carry many dyes, as opposed to a small toxin like bungarotoxin. When staining tissues, the endogenous autofluorescence of the tissue itself is often the most significant source of background. Endogenous fluorescence background in tissue is usually highest in the blue wavelengths (DAPI channel) and lowest in the far-red (Cy®5 channel). Our CF®633 bungarotoxin (catalog no. 00009) is a far-red conjugate for the Cy®5 channel with a low negative charge that should have low background from either the dye or autofluorescence.

We test fluorescent bungarotoxin on rat skeletal muscle sections. While the tissue shows autofluorescence, the bungarotoxin staining of motor endplates is usually much brighter than the background for all of the dye colors we’ve tested.  However, if you are staining human tissue (especially brain), lipofuscin autofluorescence may be bright in all channels. This usually shows up as bright, punctate dots around cell nuclei. While we would usually recommend our TrueBlack® lipofuscin quenchers for human brain tissue, they are not compatible with bungarotoxin staining. We have, however, found that EverBrite TrueBlack® Mounting Medium (cat. no. 23017) can be used to mount skeletal muscle sections stained with bungarotoxin.

Cy Dye is a registered trademark of Cytiva.

TrueBlack® Lipofuscin quencher can be used with in situ hybridization. There are several references in literature describing this (a few are listed below).

https://idp.nature.com/authorize?response_type=cookie&client_id=grover&redirect_uri=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41598-017-14484-9.

https://doi.org/10.1101/2020.02.03.931618.

https://doi.org/10.1186/s13195-019-0469-0.

If solvents or buffers containing detergents are used, TrueBlack treatment should be performed after these steps, preferably after rinsing the section with PBS. TrueBlack® may also precipitate out due to the high salt SSC wash buffers used. Rinsing the sections in PBS prior to TrueBlack treatment may help minimize this.

TrueBlack® lipofuscin quencher should be mounted using an aqueous-based mounting medium. It cannot be used with organic solvent-based mounting media like Permount™ or DPX.

The TrueBlack® lipofuscin quencher is compatible with the following mounting media (with or without DAPI):

EverBrite™ Mounting Medium
EverBrite™ Hardset Mounting Medium
Slowfade® Gold (Thermo Fisher Scientific)
Prolong® Gold (Thermo Fisher Scientific)
VECTASHIELD® (Vector Laboratories)
VECTASHIELD® Hardset (Vector Laboratories)
Fluoromount-G® (Southern Biotech)
Fluoromount™ (Sigma)

For best results, we recommend mounting samples with a glycerol-based wet-set mounting medium like EveBrite™ Mounting Medium. The quencher is also compatible with EverBrite™ Hardset Mounting Medium.

We’ve also found these mounting media to be compatible for mounting TrueBlack® Plus treated samples.
SlowFade® Gold
SlowFade® Diamond
Prolong® Diamond
Prolong® Glass
Vectashield® Hardset
Vectashield® Vybrance™
Fluoromount-G®
Mowiol®-based mounting medium

We do not recommend using the following mounting media:
Vectashield® wet-set medium
Fluoroshield™

The TrueBlack® lipofuscin quencher is hydrophobic in nature. Certain experimental conditions can cause the quencher to leave precipitates or clumps on the treated sample which can interfere with imaging. We recommend heating the vial of the stock solution of TrueBlack®, 20X in DMF to 70ºC for 5 min. to avoid this. Diluting and storing TrueBlack as a bulk 1X solution in 70% ethanol can also help.

TrueBlack® and TrueBlack® Plus are hydrophobic in nature and quench lipofuscin autofluorescence mainly through hydrophobic interactions. They could therefore stain/bind to lipid structures and quench the fluorescence signal of BODIPY and other lipid droplet stains. Also, original TrueBlack® #23007 is used in 70% EtOH, which could interfere with lipid droplet morphology and affect staining. TrueBlack® Plus #23014, on the other hand, can be used in buffer instead of 70% ethanol, and may be more suitable for combining with lipid droplet staining. However, since the binding of TrueBlack® Plus is also dependent on hydrophobic interactions, we recommend testing TrueBlack® pre-treatment and post-treatment for compatibility with lipid droplet staining in your sample type.

TrueBlack® Plus Lipofuscin Autofluorescence Quencher is cell membrane permeant, and at low concentrations it is not toxic to live cells. In live cells, the dye accumulates in lysosomes and other intracellular vesicles. Therefore, it may or may not be useful for quenching live cell autofluorescence for flow cytometry or microscopy, depending on the source and localization of the autofluorescence and target probes in the specific cell type.

The concentrations of TrueBlack® Plus that we’ve tested in live cells are much lower than those used for quenching in tissue sections, 0.001X to 0.005X (corresponding to 1:40,000 to 1:8,000 dilution of the 40X stock). Incubation can be done in cell culture medium for 30 minutes to several days, though toxicity may vary between cell types.

Our original TrueBlack® Lipofuscin Autofluorescence Quencher (catalog no. 23007)  is not soluble in aqueous buffers or media, and therefore is not compatible with live cell staining.

See below for our recommended alternatives to Akoya tyramides. Unless noted as a direct replacement, these tyramide dyes are spectrally similar alternatives. They have not been validated as direct replacements for use with Akoya kits or imaging systems.

CF® Dye Alternatives for Akoya Tyramides

Our Tyramide Amplification Kits have been demonstrated to be robust and versatile for multi-color fluorescence imaging, compatible with dye-labeled antibodies and various cell staining methods (see Figure 1).

To use a Tyramide Amplification Kit in addition to one or more dye-labeled antibodies, follow the kit protocol to fix and block samples; label with primary antibodies; then detect primary antibodies using secondary antibodies. Dye labeled secondary antibodies can be co-incubated with the HRP-conjugated secondary or HRP-streptavidin from the tyramide kit. After washing, perform the CF® Dye tyramide reaction according to the kit protocol. The tyramide reaction does not interfere with the binding of dye-labeled antibodies or other fluorescent staining reagents.

Performing multi-color detection with more than one dye tyramide on the same sample requires sequential tyramide staining reactions, followed by HRP inactivation or antibody stripping between each step. See our tech tip:

Multi-Color Fluorescence Imaging Using Biotium’s Tyramide Amplification Kits

Yes, CF® dye tyramides can be used for in situ hybridization using Tyramide Signal Amplification¹ (TSA). They are also compatible with the RNAscope assays  for RISH².

¹ doi: https://doi.org/10.1038/s41598-018-38171-5.

² doi: http://dx.doi.org/10.1101/651083.

We do not have any firsthand information about whether increasing tyramide incubation time will improve staining for thicker sections. We have tested tyramide on slide-mounted cryosections that are 10 um thick. Published protocols for 40 um floating sections recommend performing the tyramide development step for 15 minutes at room temperature.

If you are using free-floating sections, generally all incubation steps (permeabilization, antibody incubation, and washing) are longer than for sections on slides. The tyramides are small molecules and should penetrate tissue more rapidly than antibody conjugates, but you may wish to test a longer incubation time to allow the buffer to penetrate the tissue.