Lactate dehydrogenase ldh release assay

5-Aminolevulinic acid (ALA) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Necrostatin-1 (Nec-1), z-DEVD-fmk (ZDEVD) and z-IETD-fmk (ZIETD) were from Santa Cruz Biotechnology (CA). Cu-oxidised LDL (ox-LDL) and red fluorescent marked DiI-ox-LDL were both purchased from Peking Union-Biology Co. Ltd (Beijing, China). Hoechst-33342, DAPI, the ATP assay kit, the lactate dehydrogenase (LDH) release assay, and the caspase-3 and caspase-8 activity assay kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). The ApoAlert Annexin V-FITC kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Other drugs and chemicals used in this study were obtained from Sigma-Aldrich. EGS (ethylene glycol bis(succinimidyl succinate)) was obtained from Thermo Scientific.

An in vitro oxygen–glucose deprivation (OGD) model was constructed based on the method detailed by Roth et al.⁵ Briefly, the cells were cultured in an anoxic, serum‐free environment for 24 h. The cells were then returned to normal culture conditions for 18 h to simulate RIR. The ATP levels were measured by CellTiter‐Lumi™ Plus luminescent cell viability assay (Beyotime, Beijing, China). The cell membrane integrity of the R28 cells was evaluated by lactate dehydrogenase (LDH) release assay (Beyotime).