A fluorometric approach to the quantitation of cell number with application to a cell adhesion assay.
We describe a fluorometric approach to the determination of cell number. Cells are covalently labeled with fluorescein isothiocyanate (FITC) under physiologic conditions. Cells are lysed with detergent, and released fluorochrome is assessed quantitatively with a fluorescence spectrophotometer. Fluorescence varies linearly with regard to cell number over a wide range of concentrations, and allows detection of as few as 8 X 10(3) cells/ml. We describe the effect of different time and temperature incubations of FITC-labeled cells on fluorescence intensity, and we use the method to analyze the binding of peripheral blood mononuclear leukocytes to interferon-gamma-treated keratinocytes. Finally, we demonstrate that the results in an adhesion assay are comparable to those obtained with 51Cr-labeled cells. Quantitative fluorescence analysis of cell number offers a safe, inexpensive, rapid, and accurate method of determining cell number without the biological hazard and waste disposal problems associated with radioactive labeling.
D000818:Animals; D002448:Cell Adhesion; D002452:Cell Count / Q000379:methods*; D002478:Cells, Cultured; D002860:Chromium Radioisotopes; D000078404:Epidermal Cells; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D007371:Interferon-gamma; D007963:Leukocytes, Mononuclear / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D002860:Chromium Radioisotopes; D005452:Fluoresceins; D013861:Thiocyanates; D007371:Interferon-gamma; D016650:Fluorescein-5-isothiocyanate