Lactate dehydrogenase cytotoxicity assay

Cytotoxicity was assessed by lactate dehydrogenase (LDH) release from cancer cells into the culture medium. The activity of LDH in the medium was determined using a commercially available kit from Thermo Fisher (Pierce LDH Cytotoxicity Assay Kit). The assay is based on the conversion of lactate to pyruvate in the presence of LDH with parallel reduction of Nicotinamide adenine dinucleotide. The formation of Nicotinamide adenine dinucleotide hydrate from the above reaction results in a change in absorbance at 340 nm. Aliquots of media and warm reagents were mixed in a 96-well plate according to protocol instructions and absorbance was recorded using a microplate spectrophotometer system. Per cent cytotoxicity was calculated according with the formula: (Experimental Value–Effector Cells Spontaneous Control–Target Cells Spontaneous Control)/(Target Cell Maximum Control–Target Cells Spontaneous Control).