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BACKGROUND
The growth of cells can be determined by the reduction of tetrazolium salts in MTT assay. Metabolically active cells reduce the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), in part by the action of dehydrogenase enzymes, to create reducing equivalents such as NADH and NADPH. The intracellular purple formazan color results can be dissolved and quantified with spectrophotometric analysis. For the every cell type, the linear relationship between cell signal and its number produced is well established.1,2
Aim: Aim of this experiment is to study cell viability and growth by MTT assay.
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REQUIREMENTS
Sample: Cell sample
Apparatus: Microtiter plate reader with 650 nm and 570 nm filters
Multi-channel pipette
Microtiter plate (flat-bottomed)
Sterile tubes – 5 ml
Serological pipettes
Sterile pipette tips
Instruments: Inverted microscope
Incubator
Laminar flow hood
Chemicals: MTT reagent
Detergent supplement
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PROCEDURE
Cells @ 1,000-100,000 per well were plated in the cultured plate and then incubated for 6 to 24 hours. After incubation 10 μl MTT reagent per well is added followed by additional incubation for 2 to 4 hours until purple precipitate is visible. Then add 100 μl of detergent reagent and leave at room temperature in the dark for 2 hours. Finally, absorbance is taken at 570 nm in a microtitre plate.3-5
Calculation
Mean OD treated cells/ mean OD control ×100 = % of viable cells
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CONCLUSION
This experiment of MTT assay concludes that the cells are viable.
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REFERENCES
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van de Loosdrecht, A.A., et al. J. Immunol. Methods 174: 311-320, 1994.
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Ferrari, M., et al. J. Immunol. Methods 131: 165-172, 1990.
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Gerlier, D., and N. Thomasset. J. Immunol. Methods 94: 57-63, 1986.
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Alley, M.C., et al. Cancer Res. 48: 589-601, 1988.
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Mosmann, T. J. Immunol. Methods 65: 55-63, 1983.
