Lactate dehydrogenase (LDH) cytotoxicity assay

CoCl₂-induced cytotoxicity in rMC-1 cells leading to leakage of cytoplasmic enzyme lactate dehydrogenase (LDH) into culture medium was measured by Cytotoxicity Detection Kit (Roche) according to the manufacture’s instruction. CoCl₂-induced hypoxia in rMC-1 cells was performed as described above. After treatments, both cell culture medium and cell lysate were incubated with the substrate containing Diaphorase/NAD⁺ as well as INT/sodium lactate for 15 minutes. Absorbance of LDH activities were measured at 490nm. Amount of LDH released from apoptotic cells was calculated as percentage of the total LDH activities. The results were obtained from five individual experiments with duplicate samples.