Induction of splenocyte apoptosis.

Splenocytes (SCs) were submitted to γ-irradiation (40 Gy). Alternatively, SCs were cultivated in complete medium in the presence of Con-A and IL-2 for 3 days and then exposed to a lytic anti-Fas mAb (Jo2). Treated and control (untreated) cells were then washed, resuspended at the same concentration in complete medium, and kept at 37°C and 5% CO₂. After different periods of culture, the cells were stained with FITC-conjugated Annexin-V to detect apoptosis induction. The percentage of Annexin-V⁺ cells is indicated in each histogram. Upper panels represent nonirradiated control cells after 0, 2, and 6 hours of culture (A, B, and C, respectively). Intermediate panels represent γ-irradiated SCs after 0, 2, and 6 hours of culture (D, E, and F, respectively). Lower panels represent SCs after 6 and 24 hours exposure to Jo2 (G and H, respectively). Before infusion, the absence of aggregate and secondary necrotic cells (< 10%) was determined by the trypan blue exclusion method. In some experiments, cells in an advanced stage of death were assessed by using propidium iodide (PI) gating. Panel I shows control cells after 6 hours of culture (left dot plot) and γ-irradiated SCs at the time of infusion (right panel, 77% of cells are apoptotic, PI⁻ and Annexin-V⁺).