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 microplate readers capable of absorbance measurements
This protocol should not be used for:
- Fluorescence microscopy
You will need the following for this protocol:
- Cells growing in culture
- CyQUANT MTT Cell Viability Assay (Cat. No. V-13154)
- Cell culture medium
- Phosphate-buffered saline (PBS) (Cat. No. 10010-023)
- HCl, 0.01 M solution
- 96-well plates
- Microplate reader
- CO2 incubator
Protocol
1. Seed cells in a 96-well plate at a density of 104–105 cells/well in 100 µL of cell culture medium with compounds to be tested. Culture in a CO2 incubator for 24–48 hours |
2. Add 1 mL of PBS to one vial of Component A to make a 12 mM MTT stock solution; vortex or sonicate until dissolved |
3. 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 |
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
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 CyQUANT MTT Cell Viability Assay. Absorbance measurements at 570 nm were made using a microplate reader.