Figure 4

Effect of depolarizing agents on annexin V binding to apoptotic Jurkat and K562 cells. Panel A: Jurkat cells were treated with UV light; 3.5 h later, cells were assayed in one of the following buffers: buffer A; buffer B; buffer A + 1 μM gramicidin; buffer B + 1 μM gramicidin; buffer B + 1 μM valinomycin. Results are expressed as the mean fluorescence of the annexin-positive cells for each treatment relative to the mean fluorescence of cells assayed in non-depolarizing A buffer. Results are mean ± SEM for two to nine independent experiments for each treatment; all treatment/control ratios are significantly different from 1.0 by two-tailed t test (p < 0.02). Panel B: Correlation between increased annexin V binding and degree of depolarization as measured with the membrane-potential-sensitive dye DiBAC₄(3). Jurkat cells treated as described in Panel A were assayed by two-color flow cytometry with DiBAC₄(3) and AlexaFluor680-annexin V-117. DiBAC₄(3) uptake increases as membrane potential becomes less negative. Panel C: K562 cells were treated with UV light and then assayed as described in Panel A. Results are mean ± SEM of two to four independent experiments; all treatment/control ratios are significantly different from 1.0 by two-tailed t test (p < 0.02).