Viability Assay – MTT Assay

//Viability Assay – MTT Assay

Viability Assay – MTT Assay

Viability of cell is its ability to grow or stay alive. The viability assay is to check how many cells in culture are viable. Determination  of  the viability of cultured cells is important step prior to any cell-based research because only a healthy culture will show reliable and reproducible results.

  • Viability assay helps to determine the quantitative measurement of cell death. It is very crucial for any experiment involving cell lines or ex vivo cellular clinical samples.
  • In the case of pre-clinical drug discovery, potential drug candidates are usually tested against mammalian cell lines such CHO or Vero in order to assess any cytotoxic effects the compound could exert on the body’s own cells.
  • With the help of viability assay the cytotoxic effects of perturbing factor on the cellular phenotype, gene and protein expression can be investigated

 MTT Assay

The MTT assay is most common used for viability assay, the test is first described by Tim Mosmann in  1983.

MTT: MTT, is a yellow colored compound , colour is due to tetrazole which is  is reduced to formazone gives purple color in living cells. Either an acidified ethanol solution dimethyl sulfoxide or a solution of the sodium dedosylsulphate in diluted hydrochloric acid is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of coloured solution measured between 500nm to 600nm. The degree of light absorption depends on the solvent.

 

  • MTT Assay is used to check the Cytotoxicity,  which help to  determine the number of live and dead cells in a population after treatment with a pharmacological substance.
  • It is a colorimetric assay for assessing cell metabolic activity.
  • The assay uses reduction of a yellow tetrazolium salt (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, or MTT) to measure cellular metabolic activity as a proxy for cell viability. Viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT reagent to formazan, an insoluble crystalline product with a deep purple color.
  • Formazan crystals are then dissolved using a solubilizing solution and absorbance is measured at 500-600 nanometers using a plate-reader. The darker the solution, the greater the number of viable, metabolically active cells.

Performing an MTT assay is easy enough, but there can be pitfalls if one is unfamiliar with the protocol. Below is the procedure for MTT Assay

  1. Plate 1,000-100,000 cells per well in a 96-well plate and incubate with the appropriate stimulus for the desired time (usually 6-48 hours).
  2. Remove medium and wash cells with PBS.
  3. Add MTT made up in medium to a final concentration of 0.5 mg/mL.
  4. Incubate for 30 minutes to 4 hours at 37°C, until intracellular purple formazan crystals are visible under microscope.
  5. Remove MTT and add solubilizing solution (see notes below) and triturate.
  6. Incubate at room temperature or 37°C for 30 minutes to 2 hours, until cells have lysed and purple crystals have dissolved.
  7. Measure absorbance at 570 nm.

Tips and troubleshooting

Reagents

  • MTT should be kept in the dark as it is light sensitive. To avoid light MTT bottles can be covered with the foil.
  • The MTT stock must not be contaminated by bacteria or cells. The color changed of MTT indicates MTT is contaminated.  The exposure of light can also leads to change its color. The color of MTT must be yellow. To prevent MTT from contamination keep the stock in dark and at 4°C doing this will last it upto 18 months.
  • The appropriate personal protection equipment must be used while handling the MTT, as it is can cause skin and eye irritation.
  • Use DMSO, acidified isopropanol, or SDS to dissolve formazan crystals. Test to find out which works best for your experiment.

Absorbance values

  • The absorbance value taken between 500nm to 600nm should be lie between 0.75 and 1.25. In case readings are too low, the number of cells plated can be increased or the time of incubation can be increased , the cell culture conditions should be appropriate. On the other hand, plating too many cells per well will yield very high absorbance readings, as will contamination by yeast or bacteria.
  • To determine the optimal cell number per well, prepare and plate serial dilutions of cells in medium from 106 to 10³ cells/mL. This will help you decide which number of cells yields appropriate absorbance values.

 

 

NOTE:

  • Always include a positive control (untreated cells) and a blank (well containing medium only), and plate these and samples in triplicate.
  • Before adding MTT Always wash cells with PBS (step 2) in order to remove dead cells and cellular debris otherwise inaccurate results will be found.

Drawback  of the MTT assay

  • As compare to fluorescent or luminescent assays, MTT Assay is less sensitive particularly with cells that do not readily proliferate or cells with low metabolic activity.
  • It has been found that the certain chemical compounds like polyphenols, Vitamin A, coenzyme A, and DTT (dithiothreitol) interfere with the reduction of MTT to formazan and are therefore not easily compatible with MTT assays.
  • It has benn discovered exposure to MTT and formation of formazan crystals are Cytotoxic and cause damaging changes to cellular morphology.
By | 2018-05-04T06:01:46+00:00 April 30th, 2018|Molecular Biology|Comments Off on Viability Assay – MTT Assay

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