4.8. Membrane Damage Assay (LDH Assay)

Lactate dehydrogenase leakage (LDH assay) was used for determination of the membrane stability after the 3FTx treatment. In the case, 100 μL of the supernatants obtained after the centrifugation was added to the mixture of 0.5 mL 0.75 mM sodium pyruvate and 10 μL of 140 μM NADH. After incubation over 30 min at 37 °C, 0.5 mL of 0.1 M 2,4-dinitrophenylhydrazine was added to each of the samples. The absorbance of formed hydrazone was measured spectrophotometrically at 450 nm after 1 h using the microplate reader Epoch (BioTek Instruments). The amount of LDH released due to complete disruption of the cell membrane by sonification was taken as a control.