2.8. LDH cytotoxicity assay, MTT assay, and caspase-3 activity assay

Cell death was determined through LDH release assay. An LDH Cytotoxicity Assay Kit (Beyotime, China; No: C0016) was used to measure the levels of LDH released by necroptotic cardiomyocytes according to the manufacturer's manual [31]. The MTT assay was used to analyze cardiomyocyte viability. Briefly, cardiomyocytes (10³/well) were seeded into 96-well plates and were subjected to sI/R injury 24 h later. Afterwards, the MTT assay was performed and cell viability examined by measuring wells' absorbance at 490 nm on a microplate reader (Synergy™ 2; BioTek Instruments, Inc., USA) [32]. Caspase-3 activity was determined through an ELISA kit (Beyotime, China, Cat. No:C1115) according to the manufacturer's manual [33].