Tumor-induced apoptosis of Jurkat cells as assessed by the JAM assay, flow cytometry TUNEL, and loss in DiOC6 staining of mitochondria.

(A) Apoptosis of Jurkat cells as assessed by the JAM assay. Various SCCHN cell lines were coincubated with [³H]TdR-labeled Jurkat cells at a 40:1 tumor-to-lymphocyte cell ratio for 16 hours. Treatment with agonistic anti-Fas Ab (CH-11, 200 ng/mL) served as positive control for apoptosis. Target cell death was determined by measuring fragmentation of ³H-labeled target cell DNA. The error bars represent the SEM of 8 replicates. (B) Apoptosis of Jurkat cells as assessed by flow cytometry TUNEL. Fas-sensitive or Fas-resistant Jurkat cells were treated with agonistic anti-Fas Ab (CH-11, 200 ng/mL) or coincubated with PCI-13 (tumor-to-lymphocyte cell ratio of 40:1) for 16 hours. The cells were then stained with anti–CD3-PE, fixed, and stained for DNA breaks by TUNEL. TUNEL staining was assessed by flow cytometry in CD3⁺-gated cells. The percentage of TUNEL positive cells is indicated at the right corners. (C) Apoptosis of Jurkat cells as assessed by loss in DiOC6 staining of mitochondria. Fas-sensitive Jurkat cells, incubated in medium alone or with PCI-13 tumor cells (tumor-to-lymphocyte cell ratio of 40:1) for 16 hours, were stained for DiOC6 (40 nmol/L, 15 minutes, 37°C) and then, without fixation, stained by PE-conjugated anti-CD3 Ab on ice. Results shown in A, B, and C were reproduced in at least 3 different experiments.