Sensitive determination of cell number using the CyQUANT (R) cell proliferation assay

We describe here the development and characterization of the CyQUANT((R)) c ell 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 cel lular nucleic acids that can be measured using standard fluorescein excitat ion and emission wavelengths. The fluorescence emission of the dye-nucleic acid complexes correlated linearly with cell number over a large range usin g a wide variety of cell types. Under the recommended assay conditions, sta ndard 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 grow th inhibition studies with the assay were similar to those obtained in publ ished studies using other standard assays. CyQUANT assay measurements of se rum-stimulated cell proliferation correlated well with measurements made us ing [H-3]-thymidine. Also. the assay was used to analyze cellular DNA or RN A 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 re adily amenable to automation. (C) 2001 Elsevier Science B.V. All rights res erved.