© 1990 by Oxford University Press
Journal of the National Cancer Institute, Vol. 82, No. 13, 1107-1112,
July 4, 1990
© 1990 Oxford University Press
New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening
Developmental Therapeutics Program, Division of Cancer Treatment
Program Resources, Inc., National Cancer Institute, Frederick Cancer Research Facility Frederick, MD.
Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute Bethesda, MD.
*Correspondence to: Philip Skehan, M.D., Developmental Therapeutics Program, Division of Cancer Treatment, Bldg. 560, Rm. 3260, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701
We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sul-forhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.
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G. Pappa, J. Strathmann, M. Lowinger, H. Bartsch, and C. Gerhauser Quantitative combination effects between sulforaphane and 3,3'-diindolylmethane on proliferation of human colon cancer cells in vitro Carcinogenesis, July 1, 2007; 28(7): 1471 - 1477. [Abstract] [Full Text] [PDF]
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S. L. Mooberry, K. N. Weiderhold, S. Dakshanamurthy, E. Hamel, E. J. Banner, A. Kharlamova, J. Hempel, J. T. Gupton, and M. L. Brown Identification and Characterization of a New Tubulin-Binding Tetrasubstituted Brominated Pyrrole Mol. Pharmacol., July 1, 2007; 72(1): 132 - 140. [Abstract] [Full Text] [PDF]
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B. H. Morrison, J. A. Bauer, J. A. Lupica, Z. Tang, H. Schmidt, J. A. DiDonato, and D. J. Lindner Effect of Inositol Hexakisphosphate Kinase 2 on Transforming Growth Factor beta-activated Kinase 1 and NF-{kappa}B Activation J. Biol. Chem., May 25, 2007; 282(21): 15349 - 15356. [Abstract] [Full Text] [PDF]
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M. S. Choe, Z. Chen, C. M. Klass, X. Zhang, and D. M. Shin Enhancement of Docetaxel-Induced Cytotoxicity by Blocking Epidermal Growth Factor Receptor and Cyclooxygenase-2 Pathways in Squamous Cell Carcinoma of the Head and Neck Clin. Cancer Res., May 15, 2007; 13(10): 3015 - 3023. [Abstract] [Full Text] [PDF]
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G. Farnie, R. B. Clarke, K. Spence, N. Pinnock, K. Brennan, N. G. Anderson, and N. J. Bundred Novel Cell Culture Technique for Primary Ductal Carcinoma In Situ: Role of Notch and Epidermal Growth Factor Receptor Signaling Pathways J Natl Cancer Inst, April 18, 2007; 99(8): 616 - 627. [Abstract] [Full Text] [PDF]
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R. Aneja, A. M. Ghaleb, J. Zhou, V. W. Yang, and H. C. Joshi p53 and p21 Determine the Sensitivity of Noscapine-Induced Apoptosis in Colon Cancer Cells Cancer Res., April 15, 2007; 67(8): 3862 - 3870. [Abstract] [Full Text] [PDF]
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K. Schafer-Hales, J. Iaconelli, J. P. Snyder, A. Prussia, J. H. Nettles, A. El-Naggar, F. R. Khuri, P. Giannakakou, and A. I. Marcus Farnesyl transferase inhibitors impair chromosomal maintenance in cell lines and human tumors by compromising CENP-E and CENP-F function Mol. Cancer Ther., April 1, 2007; 6(4): 1317 - 1328. [Abstract] [Full Text] [PDF]
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P. T. Fok, K.-C. Huang, P. C. Holland, and J. Nalbantoglu The Coxsackie and Adenovirus Receptor Binds Microtubules and Plays a Role in Cell Migration J. Biol. Chem., March 9, 2007; 282(10): 7512 - 7521. [Abstract] [Full Text] [PDF]
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T. Frgala, O. Kalous, R. T. Proffitt, and C. P. Reynolds A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations Mol. Cancer Ther., March 1, 2007; 6(3): 886 - 897. [Abstract] [Full Text] [PDF]
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H. D. Thomas, C. R. Calabrese, M. A. Batey, S. Canan, Z. Hostomsky, S. Kyle, K. A. Maegley, D. R. Newell, D. Skalitzky, L.-Z. Wang, et al. Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for clinical trial Mol. Cancer Ther., March 1, 2007; 6(3): 945 - 956. [Abstract] [Full Text] [PDF]
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A. Hardcastle, P. Tomlin, C. Norris, J. Richards, M. Cordwell, K. Boxall, M. Rowlands, K. Jones, I. Collins, E. McDonald, et al. A duplexed phenotypic screen for the simultaneous detection of inhibitors of the molecular chaperone heat shock protein 90 and modulators of cellular acetylation Mol. Cancer Ther., March 1, 2007; 6(3): 1112 - 1122. [Abstract] [Full Text] [PDF]
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C. Tamvakopoulos, K. Dimas, Z. D. Sofianos, S. Hatziantoniou, Z. Han, Z.-L. Liu, J. H. Wyche, and P. Pantazis Metabolism and Anticancer Activity of the Curcumin Analogue, Dimethoxycurcumin Clin. Cancer Res., February 15, 2007; 13(4): 1269 - 1277. [Abstract] [Full Text] [PDF]
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M. Huang, H. Gao, Y. Chen, H. Zhu, Y. Cai, X. Zhang, Z. Miao, H. Jiang, J. Zhang, H. Shen, et al. Chimmitecan, a Novel 9-Substituted Camptothecin, with Improved Anticancer Pharmacologic Profiles In vitro and In vivo Clin. Cancer Res., February 15, 2007; 13(4): 1298 - 1307. [Abstract] [Full Text] [PDF]
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H. Zhu, M. Huang, F. Yang, Y. Chen, Z.-H. Miao, X.-H. Qian, Y.-F. Xu, Y.-X. Qin, H.-B. Luo, X. Shen, et al. R16, a novel amonafide analogue, induces apoptosis and G2-M arrest via poisoning topoisomerase II Mol. Cancer Ther., February 1, 2007; 6(2): 484 - 495. [Abstract] [Full Text] [PDF]
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N. R. Monks, S. Liu, Y. Xu, H. Yu, A. S. Bendelow, and J. A. Moscow Potent cytotoxicity of the phosphatase inhibitor microcystin LR and microcystin analogues in OATP1B1- and OATP1B3-expressing HeLa cells Mol. Cancer Ther., February 1, 2007; 6(2): 587 - 598. [Abstract] [Full Text] [PDF]
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Q. Qiu, J. Domarkas, R. Banerjee, N. Merayo, F. Brahimi, J. P. McNamee, B. F. Gibbs, and B. J. Jean-Claude The Combi-Targeting Concept: In vitro and In vivo Fragmentation of a Stable Combi-Nitrosourea Engineered to Interact with the Epidermal Growth Factor Receptor while Remaining DNA Reactive Clin. Cancer Res., January 1, 2007; 13(1): 331 - 340. [Abstract] [Full Text] [PDF]
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R. L. Kilker and M. D. Planas-Silva Cyclin D1 Is Necessary for Tamoxifen-Induced Cell Cycle Progression in Human Breast Cancer Cells Cancer Res., December 1, 2006; 66(23): 11478 - 11484. [Abstract] [Full Text] [PDF]
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K. K. W. To, Z. Zhan, and S. E. Bates Aberrant Promoter Methylation of the ABCG2 Gene in Renal Carcinoma Mol. Cell. Biol., November 15, 2006; 26(22): 8572 - 8585. [Abstract] [Full Text] [PDF]
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K. Ishida, J. C. P. de Mello, D. A. G. Cortez, B. P. D. Filho, T. Ueda-Nakamura, and C. V. Nakamura Influence of tannins from Stryphnodendron adstringens on growth and virulence factors of Candida albicans J. Antimicrob. Chemother., November 1, 2006; 58(5): 942 - 949. [Abstract] [Full Text] [PDF]
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S.-J. Cozzi, P. G. Parsons, S. M. Ogbourne, J. Pedley, and G. M. Boyle Induction of Senescence in Diterpene Ester-Treated Melanoma Cells via Protein Kinase C-Dependent Hyperactivation of the Mitogen-Activated Protein Kinase Pathway. Cancer Res., October 15, 2006; 66(20): 10083 - 10091. [Abstract] [Full Text] [PDF]
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C. P. Schroeder, H. Kadara, D. Lotan, J. K. Woo, H.-Y. Lee, W. K. Hong, and R. Lotan Involvement of Mitochondrial and Akt Signaling Pathways in Augmented Apoptosis Induced by a Combination of Low Doses of Celecoxib and N-(4-Hydroxyphenyl) Retinamide in Premalignant Human Bronchial Epithelial Cells Cancer Res., October 1, 2006; 66(19): 9762 - 9770. [Abstract] [Full Text] [PDF]
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R. G. Azrak, C. L. Frank, X. Ling, H. K. Slocum, F. Li, B. A. Foster, and Y. M. Rustum The mechanism of methylselenocysteine and docetaxel synergistic activity in prostate cancer cells. Mol. Cancer Ther., October 1, 2006; 5(10): 2540 - 2548. [Abstract] [Full Text] [PDF]
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S. Chattopadhyay, R. Zhao, E. Tsai, V. L. Schramm, and I. D. Goldman The effect of a novel transition state inhibitor of methylthioadenosine phosphorylase on pemetrexed activity. Mol. Cancer Ther., October 1, 2006; 5(10): 2549 - 2555. [Abstract] [Full Text] [PDF]
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A. M. Karst, D. L. Dai, J. Q. Cheng, and G. Li Role of p53 Up-regulated Modulator of Apoptosis and Phosphorylated Akt in Melanoma Cell Growth, Apoptosis, and Patient Survival. Cancer Res., September 15, 2006; 66(18): 9221 - 9226. [Abstract] [Full Text] [PDF]
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B. D. Zeitlin, E. Joo, Z. Dong, K. Warner, G. Wang, Z. Nikolovska-Coleska, S. Wang, and J. E. Nor Antiangiogenic Effect of TW37, a Small-Molecule Inhibitor of Bcl-2. Cancer Res., September 1, 2006; 66(17): 8698 - 8706. [Abstract] [Full Text] [PDF]
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