Jurkat cell line

The Jurkat cell line (ATCC) was grown in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated human AB-serum and 0.1% gentamicin. Apoptosis was induced in Jurkat cells by exposure to Ultraviolet-B irradiation (800 mJ/cm²) at room temperature using Stratalinker-1800 (Stratagene), as previously [26] (link). Subsequently, cells were washed twice with PBS, seeded into Petri dishes 60×15 mm in the above culture medium (0.7×10⁷ cells/mL) and incubated for 4-hrs at 37°C. The percentage of apoptotic cells was determined by flow cytometry (Facscalibur, BD) using annexin-V/propidium iodide staining (R&D). As determined in extensive preliminary experiments and verified at each preparation, after the 4-hrs incubation, approximately 60% of Jurkat cells were early apoptotic (annexin-V-positive/propidium iodide-negative, mean ±SEM: 61.2±5.0%, n = 10 experiments), with the remaining of cells being viable (negative for both annexin-V and propidium iodide, 37.5±4.3%). In such preparations, late apoptotic cells (positive for both annexin-V and propidium iodide) were typically <2% of cells (1.9±0.2%).