2.2. Human peripheral blood lymphocytes (PBLs) culture and camptothecin (CPT) treatment

The method of PBL culture and CPT treatment have been previously reported [33]. Briefly, the PBLs were isolated from the whole blood by using Ficoll (Pharmacia Biotech Inc., Piscataway, NJ) gradient centrifugation and then cultured in RPMI 1640 supplemented with 15% fetal calf serum (GIBCO BRL) and 56.25 μg/ml phytohemagglutinin (Murex Diagnostics, Norcross, GA) for 48 hours at 37°C in an incubator with 5% CO₂. We used CPT to selectively induce apoptosis of the PBLs. The baseline or spontaneous apoptosis index was obtained from the same individual’s samples that were not incubated in parallel with those treated by CPT. The dose of 250 nmol/L CPT (Cat# C9911; Sigma-Aldrich, Inc.) for the in vitro treatment of the cells for 24 hours was determined according to a previous report [22]. At the indicated time points, the aliquotted samples of cultured cells were harvested and fixed with 1% paraformaldehyde, washed with 1x PBS and finally stored in 70% ethanol at −20°C until used for the apoptotic detection by a flow cytometer.