Ldh cytoxicity detection kit

Next, we measured cell cytotoxicity by LDH Cytoxicity Detection Kit (Roche, Germany). Further, 1×10 4 cells/mL densities for 12h were plated in 24 well culture plates, then the cells were cultured by different treatment media for 24 h. LDH activity's colorimetery was measured by calculation of samples absorbance at 490 or 492 nm using an ELISA Reader (EL800; USA).

The LDH release assay is based on the measurement of lactate dehydrogenase activity in the extracellular medium. Reliability, rapidity and easy evaluation are some of the characteristics of this method (6) . The activity of LDH in the medium was determined using LDH Cytoxicity Detection Kit (Roche; Germany) which detects LDH enzyme release from damaged or dead cells. Therefore, an increase in LDH activity in each treatment shows that the treatment solution has further dead cells, i.e. it has cytotoxic effects on cells.
Briefl y, 1×10 4 cells/well of either MCF-7 or MDA-MB-231 cells was plated in 96-well culture plates for overnight and was allowed to attach for 24 h before the treatment with drugs. After that, 300 μL of fresh media containing all drug concentrations were added to each well (in triplicates) and the plates were incubated at 37 °C in 5 % CO 2 . After 24, 48 and 72 h for MCF-7 and 48 h for MDA-MB-231, the plates were removed from the incubator and then 100 μL of media from each well was carefully transferred to new plates. A volume of 100 μL of LDH substrate prepared according to the manufacturer's direction was added to each well. After 20-minute shaking at room temperature, the enzymatic reaction was stopped by adding 50 μL of 1M hydrochloric acid. Lactate dehydrogenase activity was determined by a change in absorbance at 490 nm using an ELISA reader.