Quantification of distinct patterns of apoptosis signaling in leukemia cells. Primary leukemia cells from individual patients were analyzed before (0 h) and after 16 hours in culture with or without zVAD-fmk in triplicate. In order to quantify apoptosis signaling in leukemia cells, cells were stained with antibodies against leukemia markers CD19 (or CD10) and CD34 and against cytoplasmic caspase-3 and cytochrome c. (A) Gating strategy and quantification of apoptosis events. In order to identify the leukemia cell population and to exclude debris, a gate was set on the population positive for leukemia marker (i, gate 1). In this gate, cell death (cd) was assessed by forward/side scatter criteria (ii). In the same gate (gate 1), cytochrome c release (cc) was measured simultaneously with caspase-3 activation (ac) (iii). Cells with activated caspase-3 were quantified as percent of cells in the right (top + bottom) quadrants (ac = 65% in the example shown). Cells with released cytochrome are identified by reduction of the cytochrome c signal and calculated as percent of cells in the bottom (left + right) quadrants (cc = 60% in the example shown). (B) Distinct patterns of apoptosis signaling in 3 prototype leukemia patient samples (samples 1-3). Flow cytometric plots depict cytochrome c versus caspase-3 staining in individual samples, and mean values of triplicate measurements are indicated. From these measurements, the extent of cytochrome c release (caspase-dependent, cc^dep; caspase-independent, cc^indep; total, cc^total) and of caspase-3 activation (ac^total) was calculated as described in “Patients, materials, and methods.” The following values are for examples A, B, and C: for cc^indep, –2% (t test not significant compared with control 0 h), 8% (P < .01), and 36% (P < .01), respectively; for cc^dep, 49% (P < .01), 15% (P < .01), and –2% (not significant), respectively; for cc^total (cc^indep + cc^dep), 47% (P < .01), 23% (P < .01), and 34% (P < .01), respectively; and for ac^total, 61% (P < .01), 11% (P < .01), and 17% (P < .01), respectively. Calculated differences between percentages of ac^total and cc^total (CRAC; “Results”) are +14%, –12%, and –17%, thus indicating proficient (case A) and deficient (cases B-C) apoptosis signaling in leukemia cells.