A one-step procedure for separating mouse T and B lymphocytes

✍ Scribed by C. R. Parish; Sylvia M. Kirov; Narelle Bowern; R. V. Blanden

• Publisher: John Wiley and Sons
• Year: 1974
• Tongue: English
• Weight: 907 KB
• Volume: 4
• Category: Article
• ISSN: 0014-2980
• DOI: 10.1002/eji.1830041208

✦ Synopsis

Abstract

A procedure is described for both depleting and obtaining in pure form Ig‐bearing cells from mouse lymphoid populations. The Ig‐bearing cells are identified by rosetting and the rosettes separated from the nonrosetting lymphocytes by centrifugation on Isopaque/Ficoll. The rosettes sink with the red cells and the nonrosetting lymphocytes float. The procedure is > 99.5 % efficient at depleting mouse lymphoid populations of Ig‐rosetting cells and is also > 97 % efficient at removing the Ig‐bearing cells detected by radioautography. Furthermore, after lysis of the red cells by hypotonic shock the Ig‐bearing cells are recovered in a highly pure form. The total recovery of white cells and rosettes applied is > 85 %.

This procedure was shown to produce a functional separation of T and B lymphocytes. The cell population depleted of Ig‐rosettes behaved as a pure T cell preparation. It lacked precursors of antibody‐forming cells, but contained virtually all of the Θ‐positive lymphocytes, the bulk of the helper cells detected in two in vitro hapten carrier antibody responses and all the cells which responded and produced cytotoxic cells in MLC. In contrast, the preparation of Ig rosettes expressed B cell properties. This population contained all of the antibody forming cell precursors, few helper cells and Θ‐positive lymphocytes and no MLC‐responding cells. However, there was some evidence that a small subpopulation of T cells exists which possesses surface Ig.

The separation system was used to formally demonstrate that carrier primed T cells collaborate with hapten primed B cells to generate an anti‐hapten antibody response to a hapten‐carrier conjugate. It was also established that in MLC responder B cells in no way collaborate with responder T cells to generate cytotoxic cells.