Double-marker analysis of B lymphocyte subsets using a combination of immunofluorescence and rosetting with immunomagnetic beads.
Double-marker assays were developed for enumerating CD5+ B-cells and CD25+ activated B-cells. To measure CD5+ B-cells, CD19+ (pan B-specific) cells were first isolated from peripheral blood mononuclear cells using anti-Leu12-coated immunomagnetic beads (Dynabeads). Positively selected cells were stained for surface immunoglobulin (sIg) and then incubated with anti-Leu1-coated Dynabeads. Fluorescent cells which also bound Dynabeads could be readily enumerated. The proportion of CD5+ B-cells was found to be significantly elevated in 9 of 20 patients with definite rheumatoid arthritis. B-cells bearing the interleukin-2 receptor (IL-2-R, CD25) were identified in pokeweed mitogen-stimulated PBM by sIg fluorescence and rosetting with anti-Tac-labelled Dynabeads. The kinetics of CD25 expression following activation were found to be different for B- and non-B-cells.
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D015703:Antigens, CD / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018956:CD5 Antigens; D005455:Fluorescent Antibody Technique; D006801:Humans; D007958:Leukocyte Count; D008213:Lymphocyte Activation / Q000276:immunology; D015375:Receptors, Interleukin-2 / Q000032:analysis; D012397:Rosette Formation
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018956:CD5 Antigens; D015375:Receptors, Interleukin-2