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Apoptosis, Necrosis, and Cell Viability Kits

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.

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 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.

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

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 proto-crosslinking 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).

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

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.

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.

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.


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