The MTT assay is a colorimetric assay that measures cell metabolic activity and viability. It works by measuring the reduction of yellow MTT by mitochondrial succinate dehydrogenase in living cells into an insoluble purple formazan product. The amount of formazan produced corresponds to the number of viable cells and can be quantified spectrophotometrically. The MTT assay involves adding MTT reagent to cells, incubating to allow formazan formation, solubilizing cells, and measuring absorbance to determine cell viability and proliferation rates.

1. Principle of MTT Assay: Masuma akter
This is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol- 2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the
cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple)
formazan product. The cells are then solubilised with an organic solvent (eg. Isopropanol/DMSO) and
the released, solubilised formazan reagent is measured spectrophotometrically. Since reduction of MTT
can only occur in metabolically active cells the level of activity is a measure of the viability of the cells.
The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic
events lead to apoptosis or necrosis, the reduction in cell viability. The number of assay steps has been
minimized as much as possible to expedite sample processing. The MTT Reagent yields low background
absorbance values in the absence of cells. For each cell type the linear relationship between cell number
and signal produced is established, thus allowing an accurate quantification of changes in the rate of cell
proliferation.
MTT Assay Protocol:
1. Trypsinization or scrapping the cells
2. Resuspend or splitting cells at 96 well plate
3. Check the cell confluency (if 90%-100% of the cell then it is ready for using)
4. 100~ 200 µl of desired drugs or compound in each well (usually 1 compound /drugs should have
triplicates)
5. Incubate the cells (12~24 hours)
6. Dissolve 1mg MTT in 1ml PBS solution then the molar concentration will be 2.41m M
7. in each well add 20 µl MTT reagent and rest 160 µl culture media and incubate it for 3~4 hours
8. check the cells to see the purple precipitation and the purple color is clearly visible then discard the
media carefully as it does not drain the cells.
9. after discarding media add 100 µl DMSO/ isopropanol
10. cover it with aluminum foil and mildly shake it for 10 ~15 minutes
11. Read plate in fluorescent Reader – measure OD in 570nm (background wavelength is 630nm).
MTT Molecular weight