How to Determine Cytotoxic Activity of Crude extracts by MTT assay

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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. 

  1. Culture human brain tumor cells (U251) and cervical cancer cells (HeLa) in RPMI 1640 medium and incubate them under 5% CO2 at 37° C for 48 hours to reach  80% confluence.
  2. Harvest by gentle scraping with a cell scraper and resuspended in the medium. 
  3. Dispense 100 μL of such medium with 5 × 103 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 cells.
  4. Gently aspirate supernatants and added 100μL of Dendrobium longicornu extracts over a range of four cytotoxic concentrations (100 – 800   μg/mL) prepared in medium and incubated for 24 hours.
  5. Dissolve formazan crystals which are formed after addition of extract addition in 100 μL DMSO. 
  6. Read the plate on a microplate reader (iMarkTM, Bio-Rad) at 595 nm. 
  7. Calculate the number of dead cells per well as a percentage of the control; thus, measuring cell death after drug exposure.  
  8. Plot dose-response curve for each extract to calculate the inhibition of cell growth by 50%. 
  9. Finally, calculate IC50 of extracts from the regression equation where the abscissa represents the series of the concentration of extracts and ordinate the triplicate percentage inhibition of cell growth. 

For further details, you could visit the link given below:
Paudel, M. R., Chand, M. B., Pant, B., & Pant, B. (2017). Cytotoxic Activity of Antioxidant-Riched Dendrobium longicornu. Pharmacognosy Journal, 9(4), 499–503. https://doi.org/10.5530/pj.2017.4.81

 References:
1. Mosmann T. ( 1983). Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. Journal of Immunological Methods 65(1-
2):55-63. doi:10.1016/0022-1759(83)90303-4. https://doi.org/10.1016/0022-
1759(83)90303-4.

 2. Sargent JM, Taylor CG. (1989). Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia. British Journal of Cancer 60(2):206-10. https://doi.org/10.1038/bjc.1989.252. 

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