2.9. Cell death enzyme-linked immunosorbent assay (ELISA)

DNA fragmentation indicative of apoptosis was assayed using the Cell Death Detection ELISA^PLUS kit (Roche) according to the manufacturer’s instructions, as performed previously [2]. Cardiomyocytes were washed in ice-cold PBS and harvested in cytosolic extraction buffer containing 20 mM Tris pH 7.6, 3 mM EDTA, 3 mM EGTA, 125 mM NaCl, 20 mM β-glycerophosphate and 0.4% NP-40 plus protease and phosphatase inhibitors. Samples were then spun down and the supernatant was incubated with anti-histone-biotin and anti-DNA-peroxidase in a streptavidin-coated microplate for 2 h, washed 3 times, colorimetric substrate was added, and absorbance was measured at 405 nm.