2. Annexin-V (Apoptosis assay) and activated caspase-3 measurement

Primary pre-B cells were exposed to different concentrations of DNA damage agents like mentioned in pre-B cells survival assay. After three days of culture, the cells were harvested and stained with Annexin-V according to the manufacturer (FITC Annexin V Apoptosis Detection Kit I, BD) instructions. DAPI was added at the time of measurement to identify different subsets of apoptotic cells. For caspase-3 activation assay, the cells were stained intracellularly with anti-active caspase-3 antibody according to the manufacturer (PE Active Caspase-3 Apoptosis Kit, BD) instructions.