Lactate dehydrogenase (LDH) activity assay

After AF treatment, the integrity of the cell membrane was evaluated by measuring release of intracellular LDH which reduces NAD⁺ to NADH⁺/H⁺ by oxidation of lactate to pyruvate. Two hydrogen radicals released react with tetrazolium salt to yield a red formazan salt. The LDH cytotoxicity assay was carried out according to the manufacturer’s instructions (Roche, Mannheim, Germany). Briefly, 100 μL supernatant and 100 μL reaction mixture (freshly prepared) were transferred from each well of a 96-well flat-bottomed plate. The plates were incubated for 30 min at 20 °C in the dark and absorbance was measured at 492 nm using a Polaris Microplate Reader. Blank values indicating the absorbance of the LDH were subtracted from all samples. The percent cytotoxicity was calculated according to the kit protocol.