Sensitive determination of cell number using the CyQUANT cell proliferation assay

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.