1. Mixed rosette assay for the detection of Tμ and Tγ lymphocytes
- Author
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J. Cnossen, Chris J.L.M. Meijer, G.J.M. Lafeber, and W.J.M. Damsteeg
- Subjects
education.field_of_study ,biology ,Rosette (schizont appearance) ,Lymphocyte ,Immunology ,Population ,T lymphocyte ,Peripheral blood mononuclear cell ,Molecular biology ,chemistry.chemical_compound ,Immune system ,medicine.anatomical_structure ,chemistry ,biology.protein ,medicine ,Immunology and Allergy ,Antibody ,Fluorescein isothiocyanate ,education - Abstract
A mixed rosette assay is described for simultaneous detection of T lymphocytes and lymphocytes bearing receptors for the Fc Fragments of IgM (RFcμ) or IgG (RFcγ) 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 RFcμ or RFcγ bearing lymphocytes. In peripheral blood the percentage of RFcμ bearing lymphocytes was the same whether determined directly after isolation or after overnight incubation at 37°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 RFcμ bearing lymphocytes 1 : 1, and for T lymphocytes and RFcγ bearing lymphocytes 2 : 1. In both assays the rosette suspension was preincubated for 10 min at 37°C and then centrifuged at 200 × g for 5 min. For optimal simultaneous detection of T lymphocytes and RFcμ bearing lymphocytes incubation at room temperature for at least 2 h was necessary and for T lymphocytes incuRFcγ bearing lymphocytes incubation at 4°C for at least 2 h was essential. The percentages of RFcμ or RFcγ bearing lymphocytes (Tμ and Tγ respectively) in the T lymphocyte population determined by mixed rosette assay did not differ significantly from the percentages of RFcμ and RFcγ bearing lymphocytes in lymphocyte suspensions enriched in T cells by E-rosette sedimentation. The mixed rosette assay is suitable for detection of Tμ and Tγ cells in immune deficiency and autoimmune diseases and for analysis of the mononuclear cells in lymphoreticular malignancies.
- Published
- 1980