Annexin V CF® Dye Conjugates, Azide-Free, Lyophilized

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.

Category: Apoptosis, Necrosis, and Cell Viability Kits

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 -20C, 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.

Category: Apoptosis, Necrosis, and Cell Viability Kits

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.

Category: Apoptosis, Necrosis, and Cell Viability Kits

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.

Category: Product shipping, storage, shelf life, & solubility

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.

Category: Product shipping, storage, shelf life, & solubility

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.

Category: Product shipping, storage, shelf life, & solubility