C1116

Activities of Caspase‐3, ‐8, and ‐9 were assessed using commercial kits (C1116, C1152, C1158; Beyotime) according to the manufacturer's instructions. Briefly, brain tissues were lysed and homogenized. The supernatants of the homogenates were harvested by centrifugation at 12,000 g, and the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (P0012; Beyotime). The lysates were incubated with Ac‐DEVD‐pNA (2 mmol/L, for Caspase‐3 activity), Ac‐IETD‐pNA (2 mmol/L, for Caspase‐8 activity), and Ac‐LEHD‐pNA (2 mmol/L, for Caspase‐9 activity) at 37°C for 2 h. After incubation, absorbance was read at 405 nm using a microplate reader (BioTek). Fold‐increases in activities of Caspase‐3, ‐8, and ‐9 activities were determined by comparison with control (non‐ischemic brain tissue).

Caspase 3 activity (C1116, Beyotime, China), glucose consumption (F006, Nanjing Jiancheng Bioengineering Institute, China), ATP level (S0026, Beyotime, China), and lactate production (A019, Nanjing Jiancheng Bioengineering Institute, China) were measured by different detection kits according to the manufactures’ instruction. Protein concentration was detected by BCA kit (P0011, Beyotime, China) or Bradford protein assay kit (P0006, Beyotime, China). Results were read by a microplate reader (ELX-800, BIOTEK, USA).

Cytosolic lysates of cells were prepared and normalized by a Bradford assay according to the instructions of the caspase-3 and -9 activity assay kits (C1116 and C1157, Beyotime). Ac-DEVD-pNA and Ac-LEHD-pNA were used as the substrates for caspase-3 and -9, respectively. Absorbance at OD₄₀₅ (A_{405 nm}) was determined in a Multiskan FC (Thermo Fisher Scientific) and the relative caspase activity was determined as a percentage of the value from a positive control.