Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry.
Dual parameter analysis of surface antigens in flow cytometry has become a standard method for detection of cell subsets. However, few methods have been described for the extension of multiparameter analyses to include cytoplasmic or intracellular antigens. Here we describe a simple and reproducible method for simultaneous detection of surface and intracellular antigens by flow cytometry in lysed whole blood samples. This method employs the use of digitonin, a mild glycoside detergent, and formaldehyde for permeabilization and fixation. Red blood cells are lysed with 2% acetic acid. Preparation of samples in this manner resulted in altered light scatter characteristics relative to unpermeabilized samples; however, gating issues were overcome using a combination of scatter vs. fluorescence gating. Quantitation of CD3+/CD4+ and CD3+/CD8+ cells using this method was equivalent to counts obtained with the reference method using a commercially available lysis procedure and fluorescence vs. scatter gating. The effectiveness of the permeabilization process was assessed using a monoclonal antibody designated TIA-1, which is specific for a cytoplasmic antigen associated with cytotoxic granules predominantly found in CD8+ cells. The method effectively quantitated TIA-1 positive cells and demonstrated the specificity of the reagent for a subpopulation of CD8+ lymphocytes. Using this simplified procedure for simultaneous identification of surface and cytoplasmic antigens could help in studies of cell activation, proliferation, and other functional characteristics of the immune system.
Department of Immunology/Microbiology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612.
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Landay
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Jennings
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Forman
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Raynor
D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000032:analysis*; D015703:Antigens, CD / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis*; D015704:CD4 Antigens / Q000032:analysis; D002463:Cell Membrane Permeability; D003593:Cytoplasm / Q000276:immunology*; D003594:Cytoplasmic Granules / Q000276:immunology; D005404:Fixatives / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D016131:Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology*; D012542:Scattering, Radiation
D000911:Antibodies, Monoclonal; D000941:Antigens; D015703:Antigens, CD; D000954:Antigens, Surface; D015704:CD4 Antigens; D005404:Fixatives