A "whole-blood" technique for the quantitation of canine "T-lymphocyte" progenitors using a semisolid culture system

A whole blood technique is described for the growth of concanavalin A (Con A) stimulated canine lymphocyte colonies in semisolid medium. By eliminating the routine Ficoll-Paque (F-P) gradient lymphocyte isolation, this method avoids potential problems of growth modulation due to elimination of non-lymphoid accessory cells and the influences on colony formation associated with the selective effects of F-P on lymphocyte subpopulations. Thus, the technique more closely approximates the in vivo milieu. The whole-blood method also produces higher cloning efficiencies than methods using gradient isolation of lymphocytes. Studies over a wide range of blood concentration produced a linear response of in vitro colony formation although extrapolation of the cell-dose colony-response curve did not intersect zero. Mitogen titration data indicates that a relatively large dose of Con A is required for whole blood colony formation compared to the standard F-P method. The colonies ultrastructurally were composed of lymphoblastic and lymphocytic elements which were negative for non-specific esterase activity. Characterization of cells retrieved from the colonies using rosetting techniques indicates a high percentage of the colony cells relative to canine peripheral blood cells form rosettes with human erythrocytes.