Purified CD4+ (~ 97%) and CD8+ T (~ 98%) cells were isolated from the PBMCs using anti-CD4 and anti-CD8 mAbs conjugated MACS beads. PBMCs (5 × 106 cells/ml) or purified CD4+ and CD8+ T cells (1 × 106 cells/ml) in RPMI 1640 supplemented with 10% FCS were stimulated Selleckchem CHIR-99021 with

either 5 μg/ml PHA, co-stimulated with plate bound 5 μg/ml anti-CD3 (OKT3 mAb) and 2.5 μg/ml anti-CD28 in the absence or presence of caspase inhibitors for various time periods in an atmosphere of 5% CO2 in air at 37 °C. Proliferating T cells were derived by activating purified CD4+ and CD8+ T cells with PHA for 24 h and then reseeded in media supplemented with rIL-2 (25 Units/ml). The activated T cells were cultured for 7 days prior to use. The human leukemic

T cell line, Jurkat, clone E6-1 (ATCC) were maintained in logarithmic phase of growth in RPMI 1640 supplemented with 10% FCS and 2 mM L-Glutamine in an atmosphere of 5% CO2 in air at 37 °C. To induce apoptosis, Jurkat T cells (1 × 106 cells/ml) or activated T cells (1 × 106 cells/ml) in complete medium were stimulated with recombinant Flag-tagged FasL (100 ng/ml) followed by cross-linking with anti-Flag (1 μg/ml) for 16 h. Apoptotic cells were determined using RG7422 mouse UV microscopy, annexin V staining and TMRE labelling of mitochondria as previously described (Jayaraman, 2005 and Johnson et al., 2000). Cell viability was determined by suspending treated cells in 500 μl ice-cold PBS with 10 μl of 20 μg/ml propidium iodide (PI) and the uptake of PI was analysis using flow cytometry. T cell proliferation following mitogen clonidine stimulation was determined using [3H]-thymidine incorporation. In brief, PBMCs or purified T cells were seeded at 1 × 106 cells/ml

in 96 well plates and stimulated with either PHA (5 μg/ml) or co-stimulated with anti-CD3 mAb (5 μg/ml) and anti-CD28 mAb (2.5 μg/ml) in the presence or absence of caspase inhibitors. The cells were cultured for 72 h with the last 16 h pulsed with [3H]-labelled methyl-thymidine (0.037 MBq) prior to harvest onto glass fibre filter mats using a Tomtec automated multi-well harvester (Perkin Elmer Life Sciences, Boston USA). Wallac Betaplate scintillation reagent (Perkin Elmer Life Sciences) was added to the glass fibre filter mats and the radioactivity was determined on a 1450 Microbeta liquid scintillation counter (Perkin Elmer Life Sciences, Boston USA). T lymphocyte division following mitogen stimulation was determined using CFSE labelling of the cells (Lyons and Parish, 1994). In brief, PBMCs were suspended in PBS at a density of 5 × 107/ml and incubated with 5 μM CFSE at 37 °C for 10 minutes. Following incubation with CFSE the labelled PBMCs were washed twice in RPMI to remove excess CFSE. The CFSE labelled cells were treated with mitogens as previously described in the presence or absence of caspase inhibitors.