Enumeration of IFN-gamma-producing cells by flow cytometry. Comparison with fluorescence microscopy.
A new intracytoplasmic immunofluorescence staining to detect and quantify human interferon-gamma (IFN-gamma)-producing cells by means of flow cytometry is described. Mononuclear leukocytes, stimulated in vitro to produce IFN-gamma, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic IFN-gamma was demonstrated by indirect immunofluorescence using IFN-gamma-specific mouse monoclonal antibodies. The staining exhibited a very characteristic morphology and was localized in the Golgi apparatus. An excellent agreement between the enumeration of cytoplasmic IFN-gamma-positive cells by immunofluorescence microscopy and flow cytometry was noted. However, the latter has the advantage of a standardized control, is less labor consuming and is observer independent.
D000328:Adult; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis*; D007694:Killer Cells, Natural / Q000378:metabolism; D007958:Leukocyte Count / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008860:Microscopy, Ultraviolet; D013601:T-Lymphocytes / Q000378:metabolism
D007371:Interferon-gamma