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]

Microwell tissue culture plates provide a convenient format for conducting cell growth and cellular cytotoxicity assays, but such assays often have a limited dynamic range. We have developed the digital imaging microscopy scanning system (DIM‐SCAN), which is a semiautomated fluorescence digital imaging system for quantifying relative cell numbers in situ in a variety of tissue culture plate formats, especially 96‐well plates. The system consists of an epifluorescence inverted microscope with a motorized stage, video camera, image intensifier, and an 80386 microcomputer with a PC‐Vision digitizer. Turbo Pascal software controls the stage and scans the plate taking multiple images per well, calculates total fluorescence per well, provides for daily calibration, and configures easily for a variety of tissue culture plate formats. Thresholding of digital images is used to remove background fluorescence without rinsing. Using cells pretreated with fluorescein diacetate (FDA) and loaded into 96‐well plates, linearity was seen over 3.0 logs of cell density (r = 0.996), with similar results in 6‐well (r = 0.972), 24‐well (r = 0.970), and 48‐well plates (r = 0.981). For cells stained directly in plates with Hoechst 33342, linearity was observed over 2.7 logs of cell density (r = 0.975). In low‐viability cultures stained with FDA in 96‐well plates, excessive background fluorescence completely masked viable cells, but digital thresholding dramatically reduced background fluorescence, producing a linear response (r = 9.333) over 2.7 logs of cell density (from 264 cells/well to 1.35 × 105 cells/well). DIM‐SCAN is a versatile system for quantifying total or viable cell numbers in tissue culture plates over a wide dynamic range. © 1996 Wiley‐Liss, Inc.

Determination of the doubling time for a population of cells can involve tedious calculations. We have developed computer software for MS-DOS microcomputers to expedite the analysis of tumor cell growth in vitro and in vivo. This program, DOUBLE-TIME, assists in the collection of cell numbers into a database and calculates the doubling time for a population of cells from the plot of cell growth over time. For experiments where tumor mass is measured in vivo, the software collects measurements of tumor size, calculates tumor volume (mass), generates growth curves for tumor volume change over time, and determines the doubling time of the tumor and the mean for multiple tumors. DOUBLE-TIME plots both total and viable cell numbers over time, calculates standard error of the doubling time, and the doubling time for a selected portion of a growth curve. This software also automates the cell counting process with a software-generated cell counter that allows cell counts to be tallied directly into the computer via a mouse.