Assay principle

Enzymes in the mitochondria of the cells catalyze the reduction of the yellow tetrazole dye MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) to a purple, water-insoluble formazan product. This reaction therefore reflects cellular metabolic activity. As a colorimetric assay,  absorbance of this purple dye is measured at around 570nm. There is a linear relationship between the number of metabolically active cells and absorbance, allowing us to calculate cell viability relative to untreated control.

Tetrazolium Salts

Materials

  • Cell culture facility and common cell culture supplies
  • cultured cells in their respective medium 
  • 96 well cell culture plates
  • MTT 5mg/ml in PBS
  • Isopropanol with 0.4M HCl, alternatively DMSO
  • Plate reader 

General protocol

  • seed appropriate number of cells in 100µL/well medium into 96 well plates (e.g. 10000 cells/well)
  • let the cells attach and spread over night at 37°C and 5% CO2
  • (optionally) treat cells with test compounds or test conditions and incubate for desired amount of time
    • → include replicates
    • → include negative and positive controls
  • after incubation, remove the culture supernatant and (optionally) wash the cells with PBS
  • add 100µL/well fresh culture medium containing 10µL MTT (5mg/ml in PBS, 0,22µm filter sterilized)
  • incubate 4h at 37°C and 5% CO2
  • remove cell culture supernatant
  • dissolve formazan crystals by adding 100µL/well isopropanol 0.04M HCl and place on shaker until dissolved
  • read absorbance at 570 nm using the plate reader 
  • calculate relative viability in % by dividing average absorbance of wells with treated cells by values of wells of untreated cells

Remarks 

  • many variations on this assay are possible depending on the experimental question and setup
    • → know the mechanism of action of a test drug, how long does it need to have an effect on cell viability /proliferation?
    • → test compound is interfering with absorbance measurement (because it's a dispersion, may settle down, etc)
  • Results are always interpreted relative to appropriate controls and/or internal calibration curve.
  • While you can calculate averages from triplicates or duplicate wells, for calculating errors you need to repeat experiments independently, i.e. individually diluted samples, independently seeded cells from another week of passage etc.
  • The number of metabolically active cells is proportional to the amount of produced formazan, however absorbance values get saturated at some point. Determine the linear range (and therefore appropriate number of cells) by seeding different numbers of cells, perform the assay and plot absorbance versus cell number. 
  • Since MTT reduction is caused by mitochondrial reductases, inhibitors of mitochondrial function or other reducing agents will have an influence on the signal. The same holds true for bacterial contamination. It is always recommended to visually check the cells in the microscope to help with results interpretation. 
  • MTT test can be used to determine dose-response relationships of agents that have an effect on cells. From a dose-response curve, the IC50/EC50 value can be determined. For this, different concentrations of the test compound are used (mostly in 10-fold serial dilution) and the results plotted as concentration vs. rel. viability on a logarithmic scale. The inflection point of a normalized dose-response curve corresponds to the logarithm of the inhibitor concentration that decreases the response by 50%.
  • When adding test compounds in solution, not more than 10% of the culture medium can be replaced. (max 10µL/well test compound)




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