Isolation of lymphocyte subpopulations from rabbit peripheral blood. comparison of methods based on recovery of rosette-forming cells and on recovery of Ig-bearing cells from a digestible immunoadsorbent

Abstract

Two methods to obtain lymphocyte subpopulations, defined by specific surface receptors, from rabbit peripheral blood cells were compared as to cell recovery, yield and purity of the obtained fractions: 1. a) the formation of rosettes between lymphocytes and SRBC or SRBC coated with an antigen—antibody—complement complex (D-SRBC), followed by isolation of the rosettes and recovery of the RFC, 2. b) retention of surface-Ig bearing cells on an immunoadsorbent to which antibody to rabbit Ig was covalently attached via a digestible gelatin bridge, with subsequent recovery of the retained cells by the enzymatic digestion of the bridge. Purity of the isolated cell fractions was assessed in all cases by the percentage of cells staining with FITC-labeled goat anti-rabbit Ig. Using the rosette method, all of the RFC could be recovered from rosettes and a very pure, surface-Ig negative, sub-population of cells was obtained; however, the overall number of rosettes formed (SRBC and D-SRBC) was low (8–9% of the nucleated PBC). Very good recoveries and highly enriched cell populations were obtained with the digestible immunoadsorbent, provided certain precautions to minimize cell losses were taken. Thus, 47% of the input cells, representing 90% of the lymphocytes could be recovered; separated cell populations were 96% Ig-positive or, in another experiment, 96% Ig-negative.