Cytotoxicity assay with Vero cells

Vero cells (1X10⁵ cells) were seeded in the 96 well cell culture plates along with Dulbecco Minimal Essential Medium (Gibco) with a total volume of 300 µl. Plumbagin and thymol at 10 µM and 1 µM, respectively the concentrations used in the vero cell cytotoxicity study. A volume of 3 µl and 0.3 µl was taken for 10 µM and 1 µM concentrations, respectively. The plate was incubated at 37 °C for 24 h in a humidified CO₂ incubator (5% CO2). After 24 h, 270 µl of supernatant were taken out from each well. The MTT dye was prepared to the working concentration of 5 mg/ml from the stock solution, added 10 µl to every well and incubated for 4 h at 37 °C in CO₂ incubator (Saravanan et al. 2003). Then 100 µl of DMSO was added to every well and incubated for 20 min at 37 °C in CO₂ incubator. The plate was then read in the microplate reader (Tecan Multimode reader, Switzerland) at 570 nm. All the assays were performed in triplicates.