Lactate dehydrogenase (LDH) leakage assay

The release of cytoplasmic LDH enzyme into the culture medium was determined, as previously described (13). NRK cells exposed to 100 µM CoNPs were treated with melatonin (10 or 100 µM) for 4, 24 and 48 h. Following exposure, 120 µl supernatant from the centrifuged culture media were collected (200 × g for 3 min at room temperature). The LDH activity was assayed in 3 ml reaction mixture with 100 µl pyruvic acid (2.5 mg/ml phosphate buffer) and 100 µl reduced nicotinamide adenine dinucleotide (NADH; 2.5 mg/ml phosphate buffer). The remaining volume was adjusted with phosphate buffer (0.1 M; pH 7.4). The rate of NADH oxidation was determined at a wavelength of 490 nm using a microplate reader. The amount of LDH released was expressed as LDH activity (IU/l) in culture media.