Enumeration and isolation of rabbit T and B lymphocytes by using antibody-coated erythrocytes.
Rosette formation with antibody-coated erythrocytes (Ab-E) was employed for the enumeration and isolation of rabbit B cells (Ig+T-) and T cells (Ig-T+). The cells bearing surface Ig (Ig+ cells) were enumerated by a direct immunocytoadhesion technique utilizing anti-rabbit IgG antibody-coated erythrocytes (Ab-E). To enumerate cells bearing thymus cell antigen (T+ cells), an indirect rosette technique was used in which lymphocytes were first sensitized with guinea pig anti-rabbit thymus cell antiserum and then rosetted with anti-guinea pig IgG Ab-E. To demonstrate the specificity of the anti-thymus cell antiserum, a 51Cr radioimmunoassay for counting rosettes was employed along with visual counting to enumerate Ig+ and T+ cells in lymph node cell populations. When Ig+ and T+ lymph node cells were rosetted simultaneously with sheep and human erythrocytes, no mixed rosettes (less than 1%) were observed. Ficoll-Hypaque gradient centrifugation was used to obtain purified Ig+T- and Ig-T+ cells by removing rosetted T+ and Ig+ cells, respectively. The purity of isolated Ig-T+ cells was indicated by 94 to 95% indirect rosetting with anti-thymus cell antiserum and by 0 to 3% direct rosetting with anti-rabbit IgG Ab-E. The purity of isolated Ig+T- cells was indicated by 90 t0 94% direct rosetting with anti-rabbit IgG Ab-E and by 2 to 3% indirect rosetting with anti-thymus cell antiserum. The percentage of Ig+T- and Ig-T+ cells were determined in peripheral blood and in various lymphoid organs. The isolated Ig+T- and Ig-T+ cells were also characterized by their responses to mitogens. Thus, nearly pure Ig+T- and Ig-T+ cells were isolated by "negative selection,"  which should minimize functional changes of the cells, and thereby facilitate the study of their biologic properties, e.g., their response to mitogens.
D000818:Animals; D000918:Antibody Specificity; D000961:Antilymphocyte Serum / Q000032:analysis; D001402:B-Lymphocytes; D002452:Cell Count; D002469:Cell Separation; D003601:Cytotoxicity Tests, Immunologic; D007104:Immune Adherence Reaction; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008221:Lymphoid Tissue / Q000276:immunology; D008934:Mitogens / Q000494:pharmacology; D010586:Phagocytes; D011817:Rabbits; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology
D000961:Antilymphocyte Serum; D008934:Mitogens; D011947:Receptors, Antigen, B-Cell