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Research Articles: Therapeutics, Targets, and Development

A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations

Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Childrens Hospital of Los Angeles and Division Hematology-Oncology, Department of Pediatrics, The University of Southern California Keck School of Medicine, Los Angeles, California

Requests for reprints: C. Patrick Reynolds, Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Childrens Hospital Los Angeles, MS#57, 4650 Sunset Boulevard, Los Angeles, CA 90027. Phone: 323-669-5646; Fax: 323-664-9226. E-mail: preynolds{at}chla.usc.edu

Abstract

Purpose: Cytotoxicity assays in 96-well tissue culture plates allow rapid sample handling for multicondition experiments but have a limited dynamic range. Using DIMSCAN, a fluorescence digital image system for quantifying relative cell numbers in tissue culture plates, we have developed a 96-well cytotoxicity assay with a >4-log dynamic range. Methods: To overcome background fluorescence that limits detection of viable cells with fluorescein diacetate, we used 2'4'5'6'-tetrabromofluorescein (eosin Y) to quench background fluorescence in the medium and in nonviable cells to enhance the reduction of background fluorescence achieved with digital image thresholding. The sensitivity and linearity of the new assay were tested with serial dilutions of neuroblastoma and leukemia cell lines. DIMSCAN was compared with other in vitro cytotoxicity assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and trypan blue dye exclusion. Results: Without background fluorescence reduction, scans produced a nearly flat curve across various cell concentrations from 100 to 10⁶ cells per well. Either digital image thresholding or eosin Y dramatically reduced background fluorescence, and combining them achieved a linear correlation (r > 0.9) of relative fluorescence to viable cell number over >4 logs of dynamic range, even in the presence of 4 x 10⁴ nonviable cells per well. Cytotoxicity of deferoxamine for neuroblastoma cell lines measured by the DIMSCAN assay achieved dose-response curves similar to data obtained by manual trypan blue counts or colony formation in soft agar but with a wider dynamic range. Long-term cultures documented the clonogenic ability of viable cells detected by DIMSCAN over the entire dynamic range. The cytotoxicity of two drug combinations (buthionine sulfoximine + melphalan or fenretinide + safingol) was tested using both DIMSCAN and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the wider dynamic range of DIMSCAN facilitated detection of synergistic interactions. Conclusion: DIMSCAN offers the ability to rapidly and efficiently conduct cytotoxicity assays in 96-well plates with a dynamic range of >4 logs. This assay enables rapid testing of anticancer drug combinations in microplates. [Mol Cancer Ther 2007;6(3):886–97]

Grant support: National Cancer Institute grants CA82830 and CA81403, Ashley Barrasso Foundation, and Childrens Hospital of Los Angeles Research Institute Career Development Award (T. Frgala).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Certain intellectual property rights pertaining to the contents of this article are retained by Childrens Hospital Los Angeles as described in U.S. patents 6,459,805 and 6,665,430.

Received 12/10/04; revised 12/ 9/06; accepted 2/ 1/07.

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