Ldh cytotoxicity detection kit

Cellular toxicity was determined by the LDH-Cytotoxicity Detection Kit from BioVision, which allows to directly quantify cell death in culture, based on the measurement of lactate dehydrogenase (LDH) released into growth media (15 (link)). Briefly, 3.10⁵ cells per well were to be seeded into six-well plates and incubated for 24h, before being exposed to different concentrations of rapamycin for an extra 24 h. Afterward, 50 μl of each supernatant was transferred in triplicates to a 96-well plate and supplemented with 50 μl reconstituted substrate mix. Then, the plates were incubated for 30 min at room temperature in the dark until the yellow color developed, before reading at 490 nm with a xMark microplate absorbance spectrophotometer (Bio-Rad, Mississauga, ON, Canada). Triton X-100 (1%) was used as a positive control for LDH and the negative one was obtained with untreated cells. LDH release was calculated using the following formula: % of LDH activity = [rapamycin (absorbance) − negative control (absorbance)] × 100)/[positive control (absorbance) − negative control (absorbance)]. The experiment was repeated four times.