Fig. 4. Caspase-3 and caspase-8 activity in FasL-stimulated A20 B-lymphoma cells. (A) Cells were incubated for 4 hours either without (NT) or in the presence of 100 ng/ml of FasL (FasL), and cell volumes were determined by FACS analysis by plotting forward scatter versus side scatter. After treatment with FasL, approximately 65% of cells showed a reduction in cell volume (Region 2, R2). This phenomenon was not observed in non-treated cells (Region 1, R1). (B) Caspase-3 and caspase-8 activity in situ were determined by flow cytometric analysis using the cell permeable substrates FAM-DEVD-fmk and FAM-LETD-fmk, respectively. Non-treated cells (dotted line), or cells treated with 100 ng/ml FasL from regions R1 (grey line) and R2 (black line) region, are shown. Alternatively, both cell populations R1 and R2 were separated by cell sorting. Then, either nuclear morphology was analyzed by confocal microscopy following permeabilization with methanol and PI staining (C) or DNA fragmentation was visualized following electrophoresis in agarose gels (D). In the latter experiments, cells treated with 100 ng/ml FasL (F) or left untreated (NT) served as controls. Results shown are representative of two independent experiments performed in duplicates.