Zombie Aqua die, viability assay

Zombie Aqua die, viability assay

Cell Lines and Drugs Cultured P12 and U937 cell lines were grown to confluence. Two million cells/mL were plated into a 24 well tissue culture plate with or without 20 mg/mL of mitomycin-c for 48 hrs at 37°Celsius in complete-RPMI medium + 5% fetal calf serum. Viability Staining & ViCell Counting Treated cells were washed in 10 times the volume of PBS, specifically chosen since it lacks protein, at 400g for 5  minutes. An aliquot of each cell line and treatment condition was counted on the ViCell. One million cells from each cell line and treatment condition were stained in 100 µL final volume with either 1 µL of Zombie Yellow, 1 µL of Zombie Aqua, or 20 µL of 7-AAD. All conditions were stained for 30 minutes. Then, all tubes were washed in 3 mL of growth media and spun at 400g for 5 minutes. Cell pellets were resuspended in PBS and acquired on the CytoFLEX. Results Of the three viability dyes tested, 7-AAD produced the highest signal to noise (S:N) ratio (Figure 1). Each cell line and treatment group showed similar trends in percentage viable cells with the addition of mitomycin-c or Ly-294. Additionally, each viability dye correlated to the results generated by the ViCell (Figure 2). Although the percentages of viable cells differed slightly between the viability dyes tested, it is possible that this is due to the altering mechanisms of actions of the viability dye binding.

These data suggest that it is necessary to be consistent in individual experiments with a singular viability dye of choice.

References 1- Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Roup RA, Roederer M. Amine-reactive dyes for dead cell discrimination in fixed samples. Curr. Protoc. Cytom. Chapter 9: Unit 9.34, 2010.

Reagent Details Reagent Supplier Catalog No. 7-AAD Beckman Coulter A07704 Mitomycin-C Sigma M4287 Ly-294 Sigma L9908 Zombie Yellow BioLegend 423103 Zombie Aqua BioLegend 423101