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. 2018 Apr 24;7(5):35. doi: 10.3390/cells7050035

Figure 6.

Figure 6

Detection of individual dead K562 target cells within an excess of live and dead PBMC effector cells. (A) Fixed number of 12,500 thawed cryopreserved PBMC were plated into a flat bottom 96 well plate whereby 12% of these PBMC were dead as measured by acridine-orange/PI cell viability counting. To these PBMC, a decreasing number of heat-shock-treated (HST) K562 cells were added—these K562 cells were stained with CFSE prior to the HST. Forty-seven % of these HST treated 562 cells proved to be dead by acridine-orange/PI cell viability counting. The highest number of HST-K562 cells was 1250 cells per well, corresponding to a 1:10 (0.1%) HST-K562 to PBMC ratio. For each subsequent test case, the numbers of HST K562 cells was decreased by half while keeping the PBMC numbers constant. Each serial dilution was plated in triplicate wells. In the next step PI was added, and the cells were counted by the ImmunoSpot® S6 FloroCore Reader using the TVA platform. The mean spot counts for single positive red PBMC (PI-only stained) are represented by the red symbols with SD shown for the triplicate wells. CFSE/PI double positive dead target cells were also automatically counted with the mean counts and SD represented by the orange symbols. (B) A magnified well segment is shown with the double positive cells highlighted with an arrow.