The measurement of cell-incorporated BrdU (with anti-BrdU APC) and total DNA content (with 7-AAD). Human PBMCs were stimulated with immobilized anti-human CD3 antibody, clone HIT3a, 10 μg/mL for plate coating (Catalog No. 555336), soluble anti-human CD28 antibody, clone CD28.2 at 2 μg/mL (Catalog No. 555725), recombinant human IL-2 at 10 ng/mL (Catalog No. 554603), and recombinant human IL-4 at 20 ng/mL (Catalog No. 554605) for 2 days. The cells were then washed and subsequently expanded for 3 days in culture with medium containing recombinant IL-2 and IL-4. Finally the cells were harvested and restimulated for 4 hours with PMA (Sigma, Catalog No. P-8139, 5 ng/mL) and ionomycin (Sigma, Catalog No. I-0634, 500 ng/mL). Twenty micromoles of BrdU was added for the final hour. The results correspond to the regions in the plot with R6 being apoptotic (3.31%), R4 S phase (23.5%), R3 G0/G1 (64.3%), and R5 G2 + M (6.1%).