Mixed rosette assay for the detection of T mu and T gamma lymphocytes.
A mixed rosette assay is described for simultaneous detection of T lymphocytes and lymphocytes bearing receptors for the Fc Fragments of IgM (RFc mu) or IgG (RFc gamma) in unfractionated lymphocyte suspensions. For optimal detection of T lymphocytes, treatment of sheep erythrocytes with neuraminidase was necessary. ox erythrocytes labelled with fluorescein isothiocyanate and sensitized with either rabbit IgM or rabbit IgG anti-ox antibodies served as indicator erythrocytes for RFc mu or RFc gamma bearing lymphocytes. In periphral blood the percentage of RFc mu bearing lymphocytes was the same whether determined directly after isolation or after overnight incubation at 37 degrees C. Ratios of non-fluoresceinated sheep erythrocytes to senanti-ox antibodies served as indicator erythrocytes for RFc mu or RFc gamma bearing lymphocytes. In periphral blood the percentage of RFc mu bearing lymphocytes was the same whether determined directly after isolation or after overnight incubation at 37 degrees C. Ratios of non-fluoresceinated sheep erythrocytes to senanti-ox antibodies served as indicator erythrocytes for RFc mu or RFc gamma bearing lymphocytes. In periphral blood the percentage of RFc mu bearing lymphocytes was the same whether determined directly after isolation or after overnight incubation at 37 degrees C. Ratios of non-fluoresceinated sheep erythrocytes to sensitized fluoresceinated ox erythrocytes are critical in the mixed rosette assay; for simultaneous detection of T lymphocytes and RFc mu bearing lymphocytes 1 : 1, and for T lymphocytes and RFc gamma bearing lymphocytes 2 : 1. In both assays the rosette suspension was preincubated for 10 min at 37 degrees C and then centrifuged at 200 X g for 5 min. For optimal simultaneous detection of T lymphocytes and RFc mu bearing lymphocytes incubation at room temperature for at least 2 h was necessary and for T lymphocytes and RFc gamma bearing lymphocytes incubation at room temperature for at least 2 h was necessary and for T lymphocytes and RFc gamma bearing lymphocytes incubation at 4 degrees C for at least 2 h was essential. The percentages of RFc mu or RFc gamma bearing lymphocytes (T mu and T gamma respectively) in the T lymphocyte population determined by mixed rosette assay did not differ significantly from the percentages of RFc mu and RFc gamma bearing lymphocytes in lymphocyte suspensions enriched in T cells by E-rosette sedimentation. The mixed rosette assay is suitable for detection of T mu and T gamma cells in immune deficiency and auto-immune diseases and for analysis of the mononuclear cells in lymphoreticular malignancies.
D000818:Animals; D004912:Erythrocytes / Q000201:enzymology / Q000276:immunology; D005260:Female; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D009439:Neuraminidase / Q000276:immunology; D011247:Pregnancy; D011817:Rabbits; D011961:Receptors, Fc / Q000276:immunology*; D012397:Rosette Formation / Q000379:methods; D012756:Sheep / Q000097:blood*; D013601:T-Lymphocytes / Q000145:classification / Q000276:immunology*
D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011961:Receptors, Fc; D009439:Neuraminidase