C1152

We performed the caspase 8 activity detection assay using a kit (Beyotime, C1152) following the manufacturer's instructions. Briefly, we added 100 µL of lysate to every 2 × 10⁷ cells and then transferred the lysate to a 1.5 mL centrifuge tube and lysed for 15 minutes in an ice bath. Then, we collected the cell supernatant by centrifugation (20000g, 15 minutes, 4 °C) and determined the caspase 3 activity according to a standard curve.

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).