How to Determine Cytotoxic Activity of Crude extracts by MTT assay
After total flavonoids content (TFC), total polyphenolics content (TPC), and antioxidant activity of different crude extracts of orchid including Dondrobium longicornu have been estimated, we decided to determine their cytotoxic activity using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on human brain tumor cells ( U251 ) and cervical cancer cells ( HeLa ). We employed previously established protocols (Mosmann, 1983; Sargent and Taylor, 1989) which is presented below point wisely. Culture human brain tumor cells (U251) and cervical cancer cells (HeLa) in RPMI 1640 medium and incubate them under 5% CO 2 at 37 ° C for 48 hours to reach 80% confluence. Harvest by gentle scraping with a cell scraper and resuspended in the medium. Dispense 100 μ L of such medium with 5 × 10 3 cells into each well of 96-well microtiter cell culture plate and incubated under the same conditions of cell culture for 48 hours to allow adherence and growth of cel