Fig. 4.
Fig. 4. Flow cytometric analysis of caspase activity in tumor- or Fas-stimulated T lymphocytes. / In (A) Jurkat cells were treated with anti-Fas Ab (CH-11, 200 ng/mL) for 16 hours at 37°C. In (B) Jurkat cells were cocultured on PCI-13 monolayer (tumor-to-lymphocyte cell ratio of 40:1) for 16 hours. At the end of the coincubation periods, the cells were treated with anti–CD3–fluorescein isothiocyanate followed by incubation with 10 μmol/L PhiPhiLux-G2D2 for 1 hour. CD3-positive cells were analyzed for caspase-3 cleavage activity of PhiPhiLux. Controls indicate baseline fluorescence of substrate-loaded cells in the absence of apoptotic stimulation. The percentage of positive cells for PhiPhiLux fluorescence was as follows: 5% in control; 38% in Jurkat treated with anti-Fas Ab; 31% in Jurkat coincubated with tumor cells.

Flow cytometric analysis of caspase activity in tumor- or Fas-stimulated T lymphocytes.

In (A) Jurkat cells were treated with anti-Fas Ab (CH-11, 200 ng/mL) for 16 hours at 37°C. In (B) Jurkat cells were cocultured on PCI-13 monolayer (tumor-to-lymphocyte cell ratio of 40:1) for 16 hours. At the end of the coincubation periods, the cells were treated with anti–CD3–fluorescein isothiocyanate followed by incubation with 10 μmol/L PhiPhiLux-G2D2 for 1 hour. CD3-positive cells were analyzed for caspase-3 cleavage activity of PhiPhiLux. Controls indicate baseline fluorescence of substrate-loaded cells in the absence of apoptotic stimulation. The percentage of positive cells for PhiPhiLux fluorescence was as follows: 5% in control; 38% in Jurkat treated with anti-Fas Ab; 31% in Jurkat coincubated with tumor cells.

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