title: Sensitive determination of cell number using the CyQUANT cell proliferation assay.
abstract: We describe here the development and characterization of the CyQUANT cell proliferation assay, a highly sensitive, fluorescence-based microplate assay for determining numbers of cultured cells. The assay employs CyQUANT GR dye, which produces a large fluorescence enhancement upon binding to cellular nucleic acids that can be measured using standard fluorescein excitation and emission wavelengths. The fluorescence emission of the dye-nucleic acid complexes correlated linearly with cell number over a large range using a wide variety of cell types. Under the recommended assay conditions, standard curves were linear (r(2)>0.995), detecting as few as 10-50 cells and as many as 25,000-50,000 cells with a single dye concentration, depending on cell type. Increasing the dye concentration extended the linear range of the assay to 100,000-250,000 cells. Results of cell proliferation and growth inhibition studies with the assay were similar to those obtained in published studies using other standard assays. CyQUANT assay measurements of serum-stimulated cell proliferation correlated well with measurements made using [3H]-thymidine. Also, the assay was used to analyze cellular DNA or RNA content, with the addition of a nuclease digestion step to the protocol. The assay procedure is simple and convenient, with no wash steps, and is readily amenable to automation.
authors.affiliation: Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, OR 97402, USA. laurie.jones@probes.com
authors.forename: L J
authors.initials: LJ
authors.lastname: Jones
authors.forename: M
authors.initials: M
authors.lastname: Gray
authors.forename: S T
authors.initials: ST
authors.lastname: Yue
authors.forename: R P
authors.initials: RP
authors.lastname: Haugland
authors.forename: V L
authors.initials: VL
authors.lastname: Singer
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002452:Cell Count / Q000379:methods*; D002455:Cell Division; D002478:Cells, Cultured; D003486:Cyanides; D004247:DNA / Q000032:analysis; D004285:Dogs; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D016014:Linear Models; D051379:Mice; D012313:RNA / Q000032:analysis; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D003486:Cyanides; D005456:Fluorescent Dyes; D012313:RNA; D004247:DNA