An immunofluorescent method for identifying individual IFN-gamma-producing lymphocytes.

An immunofluorescent staining method was developed for detecting human IFN-gamma-producing cells in single cell suspensions. Mononuclear leukocytes, stimulated in vitro to produce IFN-gamma, were fixed and made permeable. The cytoplasmic presence of IFN-gamma was visualized by indirect immunofluorescence using IFN-gamma-specific mouse monoclonal antibodies. The staining was found to be specific for IFN-gamma and allowed the detection of newly synthesized rather than internalized IFN-gamma molecules. The cytoplasmic fluorescence appeared locally in a polar, juxtanuclear position, which overlapped the Golgi apparatus, probably reflecting the glycosylation site of the newly formed IFN-gamma molecules. Two-colour staining experiments showed that the method is useful not only for the detection and enumeration but also for the phenotypic characterization of IFN-gamma-producing cells.