Measurement of lactate dehydrogenase (LDH) activity for necrotic cell death determination

LDH is a cytosolic enzyme that contributes to energy production within cells. Damaged and injured cells lose their membrane integrity and release LDH during necrotic cell death.26 LDH cytotoxicity assay kit was purchased from Roche Applied Sciences (Mannheim, Germany). After 24 h of exposure, the microplate was centrifuged at 100 × g (10 min). Supernatant (100 µL) was transferred to clean 96-well plates. Negative experimental controls included cells grown in medium only and cells exposed to DMSO as a vehicle control. Cells exposed to 2% Triton X-100 served as a positive control for LDH release. The catalyst solution was prepared beforehand by adding the lyophilisate provided in kit to 1 mL double distilled water (ddH₂O) and stored at 4°C. LDH reaction mixture was prepared shortly before use with 250 µL of catalyst solution and 11.25 mL dye solution. LDH reaction mixture (100 µL) was added to each well, and samples were incubated for 30 min at RT protected from light. Absorbance was read using the ELx800 Universal Microplate Reader at 490 nm. The experiment was repeated three times in replicates of three, and the data are shown as mean ± SD.