Comparison of NHS ester-based reagents to ViaKrome Fixable Viability Dye
All fixable viability dyes available are based on NHS ester activated functional groups crosslinking with primary amines on proteins. Hydrolysis competes with the crosslinking reaction, reduces the efficiency of the labeling reaction. This hydrolysis reaction is pH and temperature dependent. The half-life of the reactive group can be as short as 10 minutes at pH 8.6 at 4 °C. This means that in aqueous solutions, the dye is rendered inert over a short time period, reducing staining.
In addition, water vapor in the stock solution can compromise the performance of the reagent, reducing staining. When the staining is reduced more cells appear to be live when in reality they are dead. For this reason, reagents are typically reconstituted in DMSO, stored at -80 °C, and aliquoted in small volumes for single use. Staining procedures need to be carefully timed to obtain consistent results.
ViaKrome Fixable Viability Dyes do not use NHS ester reactive groups. The chemistries are not subject to hydrolysis, leading to more robust performance in standard assay procedures.
Assay Results 2 and 24 Hours Post Staining. A mixture of Jurkat cells and heat stressed (55 °C for 10 minutes) Jurkat cells were stained with the indicated ViaKrome Fixable Viability Dye. Samples were acquired immediately and at either 2 hours (n = 4) or 24 hours (n = 3) post staining. The dead cell population as a percentage of time zero is graphed. Error bars represent the average variation across replicates. Recruitment at 2 hours is 98-100% of time 0 and recruitment at 24 hours is 91-103% of time 0.