Why Use a Viability Dye?
Dead cells bind antibodies non-specifically skewing the percent positive calculations. Dead cells also increase in autofluorescence interfering with detection of specific signals. Gating out dead cells should be completed prior to population analysis by sequential gating.
Comparison of Sample Analysis Without and With Dead Cell Exclusion. Thawed PBMCs were stressed by heat (55 °C for 10 minutes) prior to immunostaining staining without (A) or with (B) the addition of ViaKrome 405 Fixable Viability Dye. Cells were then processed with Perfix-nc Cellular Staining Preparation Kit (Part Number B10825) and stained with Granzyme B-FITC, CD19-PE, CD14-ECD, CD79a-PC5.5, CD3-PC7 and CD45-Krome Orange. Data was acquired using the CytoFLEX LX N-V-B-Y-R-I series flow cytometer and analyzed with Kaluza Analysis software. Gate statistics show the percent of the parent population and differ in the two conditions showing the effect of eliminating dead cells prior to sequential gating.