CCK-8 assay and LDH release assay

A CCK-8 assay measured the cell viability, according to the manufacturer's protocols. PC12 cells were cultured in 96-well plates (1×10⁴ cells/well) and, following OGD/R, CCK-8 (10 µl per well) was added, and cells were incubated for 4 h at 37°C. The OD value at the wavelength of 450 nm was detected by an enzyme labeling instrument.

Cytotoxicity was assessed by measuring the level of LDH in the culture medium. Following reperfusion, the medium was collected for LDH level measurement. The LDH level was measured by spectrophotometry determination at 440 nm, according to the manufacturer's protocol. The release of LDH reflects the degree of cell damage.