Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Sulforhodamine B colorimetric assay for cytotoxicity screening


The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1


  1. Skehan, P. et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107–1112 (1990).

    Article  CAS  Google Scholar 

  2. Lillie, R.H.J. Conn's Biological Stains 9th edn (Williams & Wilkins, Baltimore, 1977).

    Google Scholar 

  3. McCaffrey, T., Agarwal, L. & Weksler, B. A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro. In Vitro Cell Dev. Biol. 24, 247–252 (1988).

    Article  CAS  Google Scholar 

  4. Monks, A. et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J. Natl. Cancer Inst. 83, 757–766 (1991).

    Article  CAS  Google Scholar 

  5. McLaren, C., Ellis, M. & Hunter, G. A colorimetric assay for the measurement of the sensitivity of herpes simplex viruses to antiviral agents. Antiviral Res. 3, 223–234 (1983).

    Article  CAS  Google Scholar 

  6. Kleymann, G. & Werling, H. A generally applicable, high-throughput screening-compatible assay to identify, evaluate, and optimize antimicrobial agents for drug therapy. J. Biomol. Screen 9, 578–587 (2004).

    Article  CAS  Google Scholar 

  7. Pittayakhajonwut, P. et al. An anti-herpes simplex virus-type 1 agent from Xylaria mellisii (BCC1005). Tetrahedron Lett. 46, 1341–1344 (2005).

    Article  CAS  Google Scholar 

  8. Griffon, G., Merlin, J. & Marchal, C. Comparison of sulforhodamine B, tetrazolium and clonogenic assays for in vitro radiosensitivity testing in human ovarian cell lines. Anticancer Drugs 6, 115–123 (1995).

    Article  CAS  Google Scholar 

  9. Pauwels, B. et al. Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies. Cancer Chemother. Pharmacol. 51, 221–226 (2003).

    CAS  PubMed  Google Scholar 

  10. Plumb, J., Milroy, R. & Kaye, S. Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res. 49, 4435–4440 (1989).

    CAS  PubMed  Google Scholar 

  11. Rubinstein, L.V. et al. Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. J. Natl. Cancer Inst. 82, 1113–1117 (1990).

    Article  CAS  Google Scholar 

  12. Haselsberger, K., Peterson, D., Thomas, D. & Darling, J. Assay of anticancer drugs in tissue culture: comparison of a tetrazolium-based assay and a protein binding dye assay in short-term cultures derived form human malignant glioma. Anticancer Drugs 7, 331–338 (1996).

    Article  CAS  Google Scholar 

  13. Perez, R., Godwin, A., Handel, L. & Hamilton, T. A comparison of clonogenic, microtetrazolium and sulforhodamine B assays for determination of cisplatin cytotoxicity in human ovarian carcinoma cell lines. Eur. J. Cancer 29A, 395–399 (1993).

    Article  CAS  Google Scholar 

  14. Keepers, Y. et al. Comparison of the sulforhodamine B protein and tetrazolium (MTT) assays for in vitro chemosensitivity testing. Eur. J. Cancer 27, 897–900 (1991).

    Article  CAS  Google Scholar 

  15. Huang, H., Shu, S., Shih, J., Kuo, C. & Chiu, I. Antimony trichloride induces DNA damage and apoptosis in mammalian cells. Toxicology 129, 113–123 (1998).

    Article  CAS  Google Scholar 

Download references


We would like to thank Y. Thebtaranonth for his invaluable input.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Vanicha Vichai.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Vichai, V., Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1, 1112–1116 (2006).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing