Evaluation of six fluorescent protein stains for use in flow microfluorometry

Flow microfluorometric (FMF) analysis of stained cells has provided protein distribution histograms for large populations of cells. Spectral data and staining protocols were evaluated for six fluorescent protein dyes suggested for staining cells in liquid suspension. The requirements for dyes and/or staining protocol included minimal cell clumping and cell loss, near-optimal dye excitation at existing laser wavelengths, and tenacity of the dye/protein interaction. These criteria were best satisfied by fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RITC). Both fluorescamine and 8-aniline-1-naphthalene sulfonic acid (ANSA) showed potential applicability for use in systems where excitation wavelengths in the ultraviolet range are available. Protein staining with fluorescamine was extremely rapid. Brilliant sulfaflavine and 1-dimethyl-aminonaphthalene-5-sulfonyl chloride (DANSYL) were found unsatisfactory in these studies, since the former dye tended to diffuse from the cells, while the latter induced excessive cell clumping and cell loss. These techniques have application to immunofluorescence analysis and can also be profitably employed in dual-parameter analysis systems in connection with double-staining techniques for simultaneous DNA and protein analysis.