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


CONCLUSIONS
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.
Frgala
Tomas
T
Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Children's Hospital Los Angeles, MS#57, 4650 Sunset Boulevard, Los Angeles, CA 90027, USA.
Kalous
Ondrej
O
Proffitt
Robert T
RT
Reynolds
C Patrick
CP
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects*; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004801:Eosine Yellowish-(YS) / Q000494:pharmacology; D017313:Fenretinide / Q000008:administration & dosage; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000473:pathology; D008558:Melphalan / Q000008:administration & dosage; D008856:Microscopy, Fluorescence / Q000379:methods*; D009447:Neuroblastoma / Q000188:drug therapy* / Q000473:pathology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D013110:Sphingosine / Q000008:administration & dosage / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D017313:Fenretinide; D019328:Buthionine Sulfoximine; D011493:Protein Kinase C; D013110:Sphingosine; C005682:safingol; D008558:Melphalan; D004801:Eosine Yellowish-(YS)