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.
Landay
A
A
Department of Immunology/Microbiology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612.
Jennings
C
C
Forman
M
M
Raynor
R
R
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