Lactate Dehydrogenase (LDH) Assay

Cancer cell membrane integrity was analyzed for the detection of cell death using the LDH cytotoxicity assay kit (Thermo Scientific) based on the manufacturer instructions. Briefly, cancer cells were seeded at 0.15x10⁶ cells/ml in 100 µl volume in a 96-well tissue culture dish in two sets of triplicate wells. The control set was for maximum LDH release by subjecting cells to undergo complete lysis, and the test set was for spontaneous LDH release. Cells were subjected to incubation and subsequent media change as described for generating serum free TCM. Three days after initial seeding, 10 µl of 10x lysis buffer and 10 µl of sterile water were added to the control and test set, respectively. After incubating the plate for 45 min in the incubator at 37°C/5% CO₂, 50 µl from each well was transferred to a fresh 96 well plate, mixed with 50 µl LDH reaction mix, and incubated for additional 30 min at RT protected from light. Finally, the reaction was stopped by adding the stop solution and absorbance was measured at 490nm and 680nm by a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices). Absorbance was corrected by subtracting background signal of the instrument followed by correcting for the absorbance values of corresponding media. The percentage of maximum LDH release was calculated with respect to corresponding control set.