title: Determination of co-expression of activation antigens on proliferating CD4+, CD4+ CD8+ and CD8+ lymphocyte subsets by dual parameter flow cytometry.
abstract: The incidence of activation markers on proliferating CD4+, CD4+ CD8+ and CD8+ lymphocyte subsets was determined in a single laser Epics-C fluorescence-activated cell sorter system, using a series of double staining combinations. Experiments were performed after 3 days of culture with PHA on cell fractions enriched for CD4+ or CD8+ lymphocytes before initiation of culture. The percentage of CD4+, CD4+ CD8+ and CD8+ lymphocytes in the total population was determined using double staining with Leu3 PE for the detection of CD4+ cells, and Leu2 FITC for the detection of CD8+ cells. Next, double stainings with Leu3 and Leu2 antibodies conjugated with PE and antibodies directed against activation markers (M) IL-2 receptor, transferrin receptor, HLA-DR antigen and CALLA conjugated with FITC were performed, using the following combinations: Leu3 and Leu2/M, Leu3/M and Leu2/M. The expression of activation markers on CD4+ CD8+ lymphocytes was calculated from the results. Our findings indicate that CALLA is expressed on most CD4+ and all CD4+ CD8+ cells, and on a small percentage of CD8+ lymphocytes; the IL-2 receptor was expressed on most CD4+ cells, on approximately three-quarters of CD4+ CD8+ cells and half the CD8+ cells; HLA-DR was expressed on a small percentage of CD4+ cells, all CD4+ CD8+ cells and half of CD8+ cells. The transferrin receptor was almost exclusively expressed on CD4+ CD8+ cells. The standard deviation of the calculated values did not exceed 13% and this analysis can generally be applied to determine the co-expression of a third marker in a mixture of single and double stained cells using conventional methods.
mesh_terms: D000328:Adult; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006684:HLA-DR Antigens / Q000032:analysis; D006801:Humans; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000145:classification / Q000276:immunology*; D008875:Middle Aged; D015260:Neprilysin
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D006684:HLA-DR Antigens; D015260:Neprilysin