A single laser flow cytometry method to evaluate the binding of three antibodies.
Here we describe a flow cytometry method which permits the collection of data regarding three staining parameters of a single cell from a suitable combination of dual parameter stainings using PE- and FITC-labelled mAbs and a single argon laser. The technique neither requires a third dye with non-overlapping emission spectra nor a second light source. In order to test the accuracy of this method, we compared data calculated using this triple parameter analysis with data obtained by double staining a presorted population homogeneously positive for one parameter. In experiments using either resting or in vitro activated T cells, the percentages obtained with both methods were identical (P greater than 0.05). Using this method we tested which T cell sub-subpopulation is responsible for the weak CD25 (IL-2R alpha) expression constantly associated with freshly isolated human T cells and concluded that it is predominantly expressed on the CD4+ CD45RO+ (CD4 positive memory) T cell subpopulation.
Maurer
D
D
Institute of Immunology, University of Vienna, Austria.
Felzmann
T
T
Knapp
W
W
D000328:Adult; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis* / Q000276:immunology; D000943:Antigens, Differentiation / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006649:Histocompatibility Antigens / Q000032:analysis; D006801:Humans; D007834:Lasers; D017493:Leukocyte Common Antigens; D008213:Lymphocyte Activation / Q000276:immunology; D010799:Phycoerythrin; D010835:Phytohemagglutinins / Q000494:pharmacology; D015375:Receptors, Interleukin-2 / Q000032:analysis; D016176:T-Lymphocyte Subsets / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D013861:Thiocyanates
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; D000945:Antigens, Differentiation, T-Lymphocyte; D015704:CD4 Antigens; D016827:CD8 Antigens; D005452:Fluoresceins; D006649:Histocompatibility Antigens; D010835:Phytohemagglutinins; D015375:Receptors, Interleukin-2; D013861:Thiocyanates; D010799:Phycoerythrin; D017493:Leukocyte Common Antigens; D016650:Fluorescein-5-isothiocyanate