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Sulforhodamine B colorimetric assay for cytotoxicity screening

Nature Protocols volume 1, pages 11121116 (2006) | Download Citation



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.

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  1. 1.

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

  2. 2.

    Conn's Biological Stains 9th edn (Williams & Wilkins, Baltimore, 1977).

  3. 3.

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

  4. 4.

    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).

  5. 5.

    , & A colorimetric assay for the measurement of the sensitivity of herpes simplex viruses to antiviral agents. Antiviral Res. 3, 223–234 (1983).

  6. 6.

    & 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).

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

    , & 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).

  11. 11.

    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).

  12. 12.

    , , & 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).

  13. 13.

    , , & 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).

  14. 14.

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

  15. 15.

    , , , & Antimony trichloride induces DNA damage and apoptosis in mammalian cells. Toxicology 129, 113–123 (1998).

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

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  1. National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Patumtani 12120, Thailand.

    • Vanicha Vichai
    •  & Kanyawim Kirtikara


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The authors declare no competing financial interests.

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Correspondence to Vanicha Vichai.

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