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Metabolic activity—based cell viability assay

MTT is a colorimetric assay used to assess cell viability as a function of cell number based on metabolic activity. The water-soluble MTT is converted to an insoluble formazan, which is then solubilized and the concentration determined by optical density at 570 nm.

This protocol can be used for:

  • Quantification of cell number based on metabolic activity in microplates

This protocol should not be used for:

  • Fluorescence microscopy

You will need the following for this protocol:

Protocol

1. Add 1 mL of PBS to one vial of Component A to make a 12 mM MTT stock solution; vortex or sonicate until dissolved
2. Add 10 mL of 0.01 M HCl to one tube of SDS (Component B). Mix by inversion or sonication until the SDS dissolves. Once prepared, the solution should be used promptly
3. Seed cells in a 96-well plate at a density of 104–105 cells/well in 100 µL of culture medium with compounds to be tested. Culture in a CO2 incubator for 24–48 hours
4. Wash the cells by removing the medium and replacing it with 100 µL of fresh medium
5. Add 10 µL of the MTT stock solution to each well
6. Incubate at 37°C for 4 hours at 37°C in a CO2 incubator
7. Add 100 µL of the SDS-HCl solution to each well
8. Incubate at 37°C for 4 hours at 37°C in a CO2 incubator
9. Mix each sample by pipetting up and down
10. Read absorbance at 570 nm

 

Protocol tips

  • MTT dissolves more quickly if the culture medium is warmed to 37°C
  • Washing is not required after staining
  • Remaining MTT solution can be stored for four weeks at 4°C
  • Add 10 µL of the MTT stock solution to 100 µL of medium alone for a negative control well
  • A cell titration experiment using a range of cells from 103–105 cells/well can be used to generate a standard curve

Line graph showing quantification of cell numbers of Jurkat cells using the Vybrant® MTT Cell Proliferation Assay Kit
Jurkat cells were diluted to the indicated cell numbers in 100 µL volumes, delivered to the wells of a microplate, and incubated for 4 hours with the Vybrant® MTT Cell Proliferation Assay Kit. Absorbance measurements at 570 nm were made using a microplate reader.