Method and Results Two common cell lines, U937 and Jurkat (available through ATCC), were used. Initial cultures for each line were seeded at both a high and low density, so that the final cell concentration at analysis would be dense or light, respectively. After one week of culture, cells were resuspended and 1 mL transferred directly from each tissue culture flask into a 1.5 mL tube. 7-Amino-actinomycin D (7-AAD, Cayman Chemical), a fluorescent dye which is excluded by viable cells, was used as a marker of cells with compromised outer membranes.
Five (5.0) µL of a 1 mg/mL stock 7-AAD solution, along with 50 µL of AccuCount Fluorescent Particles (Spherotech; ACFP-50-5) were added to each sample tube and mixed thoroughly. Tubes were kept at room temperature (RT) in the dark, and sampled between 5 and 30 minutes after addition of 7-AAD, with gentle mixing immediately prior to analysis.
A hemacytometer was used to perform the microscopic cell counts. Appropriate dilutions of cell samples were made into phosphate-buffered saline containing Trypan Blue. Counts were performed in triplicate. At least 100 nonblue cells were counted for each replicate.
With the C6, only a single 2-D density plot of forward light scatter (FSC-A) versus 7-AAD fluorescence (7-AAD FL3-A) was required for data analysis. A negative control sample for each cell type (no added 7-AAD) was used to define the viable-cell gate (Figure 2A, gate P5). This gate included events with high FSC-A, and defined the FL3-A background fluorescence. The dead-cell gate (Figure 2B, gate P6) was set using a cell sample containing 7-AAD. This gate only needs to be set once for each cell type within a given experiment.
Using the Statistics Tab in CFlow, the events/µL for these two gates were displayed for triplicate samples (Figure 2C). This data was copied and pasted into a spreadsheet program to determine the standard deviation (SD) and coefficient of variation (CV) for triplicate measurements (Figure 2D).