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Enumeration and Isolation of Rabbit T and B Lymphocytes by Using Antibody-Coated Erythrocytes¹

From the Department of Microbiology, University of Illinois at the Medical Center, Chicago, Illinois

Abstract

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 to 94% direct rosetting with anti-rabbit IgG Ab-E and by 2 to 3% indirect rosetting with anti-thymus cell antiserum. The percentages 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.

Footnotes

¹ This work was supported by United States Public Health Service Grant AI-07043.

² In partial fulfillment of the requirements for the Doctor of Philosophy degree in the Graduate College, University of Illinois at the Medical Center, Chicago, Illinois.

³ Correspondence and reprints to: Sheldon Dray, M.D., Ph.D., Department of Microbiology, University of Illinois at the Medical Center, 835 South Wolcott, Chicago, Illinois 60612.