Flow cytometric method for simultaneous detection of lymphocyte-K562 conjugates and immunophenotyping of the conjugate forming cells.
A flow cytometric method for the simultaneous quantification and immunophenotyping of conjugates formed by human peripheral blood lymphocytes (PBL) and K562 cells has been developed. The method uses three fluorescent probes. One of the fluorescent probes (F-18) is used for labeling of PBL prior to incubation with K562 cells. After incubation the cells are treated with monoclonal antibodies labeled with phycoerythrin and Red613, respectively. The combination of F-18 fluorescence and light scattering signals enables identification and quantification of the conjugates while the fluorescence of the monoclonal antibodies provides information about the phenotype of the conjugate forming cells. Results obtained using different monoclonal antibodies are presented. The highest conjugate forming capacity has been found in the CD56+CD8+ population while the CD4+CD8- population has shown the lowest capacity to form conjugates. The influence of a washing step on the conjugate formation is discussed. The possibility to use the method in combination with a cytotoxicity assay is indicated.
Radosević
K
K
University of Twente, Department of Applied Physics, Enschede, The Netherlands.
de Grooth
B G
BG
Greve
J
J
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D015704:CD4 Antigens / Q000032:analysis; D019002:CD56 Antigen; D016827:CD8 Antigens / Q000032:analysis; D002449:Cell Aggregation; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D016130:Immunophenotyping; D007942:Leukemia, Experimental / Q000378:metabolism / Q000473:pathology*; D007951:Leukemia, Myeloid / Q000378:metabolism / Q000473:pathology*; D008214:Lymphocytes / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism; D010799:Phycoerythrin; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D015704:CD4 Antigens; D019002:CD56 Antigen; D016827:CD8 Antigens; D005456:Fluorescent Dyes; D010799:Phycoerythrin