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Metabolic activity—based cell viability assay

XTT is a colorimetric assay used to assess cell viability as a function of cell number based on metabolic activity. This rapid, sensitive, non-radioactive assay is detected using standard microplate absorbance readers.

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:


1. Grow 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
2. Make a 10 mM PMS solution in phosphate-buffered saline (3 mg PMS into 1 mL PBS)
3. Dissolve 4 mg of XTT in 4 mL of 37°C cell culture medium
4. Add 10 µL of the PMS solution the 4 mL of XTT solution created in step 3 immediately before labeling cells
5. Add 25 µL of XTT/PMS solution directly to each well containing 100 µL cell culture
6. Incubate for 2 hours at 37°C in a CO2 incubator
7. Read absorbance at 450 nm


Protocol tips

  • XTT dissolves more quickly if the culture medium is warmed to 37°C
  • Washing is not required after staining
  • Store remaining PMS solution at –20°C
  • A cell titration experiment using a range of cells from 103–105 cells/well can be used to generate a standard curve