MTT Assay of Cell Numbers after Drug/Toxin Treatment   

Abstract

Materials and Reagents

1. Raw264.7, MCF-7 or Hela cells
   
2. Thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: M5655 )
   
3. DMSO
   
4. DPBS (Life Technologies, Invitrogen^TM, catalog number: 14190-250 )
   
5. General chemicals (Sigma-Aldrich)
   

Equipment

1. 96 well plate
   
2. Shaking table
   
3. Paper towels
   
4. Incubator
   

Procedure

1. Plate 500-10,000 cells in 200 μl media per well in a 96 well plate. Leave 8 wells empty for blank controls.
   
2. Incubate (37 °C, 5% CO₂) overnight to allow the cells to attach to the wells.
   
3. Add 2 μl of drug of interest dissolved in DMSO to each well. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the samples into the media.
   
4. Incubate (37 °C, 5% CO₂) for 1-5 days to allow the drug/toxin to take effect.
   
5. Make 2 ml or more of MTT solution per 96 well plate at 5 mg/ml in DPBS. Do not make a stock as MTT in solution is not stable long-term.
   
6. Add 20 μl MTT solution to each well. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the MTT into the media.
   
7. Incubate (37 °C, 5% CO₂) for 1-5 h to allow the MTT to be metabolized.
   
8. Dump off the media (dry plate on paper towels to remove residue if necessary).
   
9. Resuspend formazan (MTT metabolic product) in 200 μl DMSO. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the formazan into the solvent.
   
10. Read optical density at 560 nm and subtract background at 670 nm. Optical density should be directly correlated with cell quantity.
    

Acknowledgments

This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. This protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA [Chen et al. (unpublished)].

References

1. Hayon, T., Dvilansky, A., Shpilberg, O. and Nathan, I. (2003). Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leuk Lymphoma 44(11): 1957-1962.