Modulation of intracellular calcium with flavopiridol treatment. (A) Calcium flux in isolated CD19⁺ cells from 5 individual patients was measured using fluorescent confocal microscopy. Cells were incubated with or without 1.5 μM FP for 1, 3, or 5 hours, then transferred to calcium-free buffer and plated on polylysine-coated coverslips for examination. Data represent a mean fluorescent intensity of all cells in 2 randomly selected fields. Ionomycin was used as a positive control for calcium release from intracellular stores (not shown). Data are shown as fold increases in fluorescence intensity of treated cells relative to the untreated controls. These same samples were concurrently analyzed for (B) annexin binding and (C) mitochondrial depolarization. (D) Effect of EGTA on apoptosis and mitochondrial membrane potential of FP-treated CLL patient cells. CLL cells (N = 6) were preincubated with 2 mM EGTA for 1 hour prior to adding 1.5 μM FP for an additional 6 hours. Annexin binding, PI uptake, and JC-1 aggregation were assessed by flow cytometry. Data are shown relative to the untreated sample at the same time point. By itself, EGTA did not impact annexin binding or mitochondrial depolarization (P = .85 and P = .17, respectively). Differences in FP-induced annexin binding with and without EGTA did not reach significance (P = .07). FP-mediated mitochondrial depolarization was unaffected by the addition of EGTA (P = .59). EGTA at 4 mM produced similar results (data not shown).