LDH assay for cytotoxicity

To determine the maximum non-toxic concentration of E. sativa extracts and fractions, C2C12 myotubes, H4IIE hepatocytes and 3T3-L1 cells cultured in 12-well plates were treated with the extracts in concentrations ranging from 7.25 to 200 μg/mL or with the vehicle control (0.1% DMSO) for 18 h. At the end of the treatment, the cell culture media were removed and kept on ice for further analysis. The cells were then rinsed with phosphate buffer saline (PBS) and lysed with 1% Triton X-100, for 10 min. The activity of LDH in media (released LDH) and lysates (cellular LDH) was assessed using cytotoxicity detection LDH kit (Roche, Mannheim, Germany). The cytotoxicity was expressed as the ratio of released LDH to total LDH (total LDH = released LDH + cellular LDH) and was used to determine the maximum non-toxic concentration for each extract. Experiments were performed in triplicate.