Cy3 coupled streptavidin

TUNEL assay: Detection of DNA fragmentation, a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was performed by following the protocol of the TUNEL assay kit (Roche). Briefly, fixed cells or tissue samples were permeabilized with 0.2% TX-100 in PBS (30 min at room temperature), washed with PBS, incubated with 300 U ml^−l TUNEL enzyme and 6 μmol l⁻¹ biotinylated deoxyuridine triphosphate (Roche Diagnostics, Mannheim, Germany). The extremities of the biotin coupled DNA were revealed by using Cy-3-coupled streptavidin (1:1,000 in PBS, Jackson Immunoresearch). The slides were washed with PBS, DAPI-stained, then washed with PBS and finally, mounted with Fluoromount G (SouthernBiothec). Images were acquired with Zeiss Axiovision fluorescence microscopy and NIS element AR 4.20.01 Nikon fluorescence microscopy. Caspase-3 activity assay: Cells were first harvested by scraping. Cell pellets were obtained by centrifugation at 4 °C and lysed. The caspase 3 activity assay was performed according to the manufacturer’s instructions (Biovision caspase-3 colorimetric assay kit). Total protein concentrations were measured with the BCA assay kit using BSA as a standard (Pierce Biotechnology, Rockford, IL, USA). Absorbance readings were done on a TECAN infinite F500.