To discriminate necrotic/late apoptotic from early apoptotic cells, the presence of PS on the outer surface of the apoptotic cells was detected from fluorescein isothiocyanate (FITC)-conjugated annexin V binding to PS at the cell surface (14), and necrosis/late apoptosis was assessed from the amount of propidium iodide (PI)-positive cells. Briefly, 106 splenic leukocytes were harvested and washed twice with annexin washing buffer (AWB, 10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2). The cell pellet was resuspended in 100 µl of annexin-V/PI labelling solution (eBioscience, USA), and incubated for 15 min at room temperature. After washing with AWB, CD45-positive lymphocytes were analysed by flowcytometry.