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

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 10 6 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 Â 10 4 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]

Activity of MM-398, Nanoliposomal Irinotecan (nal-IRI), in Ewing's Family Tumor Xenografts Is Associated with High Exposure of Tumor to Drug and High SLFN11 Expression

“…We assessed cytotoxicity of SN-38 using the DIMSCAN cytotoxicity assay as previously described (31,32) and SLFN11 expression by Taqman real-time reverse transcription-PCR (RT-PCR) as previously described (33) Table S1). Relative IC 50 (rIC 50 ) was calculated based on NCI Pediatric Preclinical Testing Program (PPTP) guidelines (32).…”

Cellular Chemosensitivity Assays: An Overview

“…Although it proved as accurate and sensitive as fluorescence based viability assays (47), the clonogenic cell survival assay lacks the dynamic range of newer fluorescent methods or the ATP assay (48). The conventional CSA also cannot measure impact of cell-cell communication on cell proliferation, because cells are plated at low densities to form colonies.…”

Study on potential antitumor mechanism of a novel Schiff Base copper(II) complex: Synthesis, crystal structure, DNA binding, cytotoxicity and apoptosis induction activity

“…Clonogenic survival was defined as the ability of the cells to maintain their clonogenic capacity and to form colonies [52,53]. 300 cells were seeded into 6-well plate to form clones with varying amounts of complex 1 (0-80 μM) were each added to the plate in the 3rd day, in a humidified atmosphere containing 5% CO 2 at 37°C.…”