Enhancement of mitogen-induced lymphocyte proliferation in sheep

Conditions were established to optimize the lymphocyte proliferation test in sheep, a species where proliferation under standard conditions is lower than for other species. Optimum proliferation of phytohemagglutinin-stimulated lymphocytes was achieved in TCM-199 supplemented with 5% (v/v) horse serum, 0.2 mM sodium pyruvate and 5 x 10(-5) M beta-mercaptoethanol. Proliferative responses in this medium were 2.5-fold higher or more than when using a standard medium (RPMI-1640 + 10% heat-inactivated fetal bovine serum). Lymphocytes cultured in medium containing horse serum also responded to concanavalin A as well as or better than lymphocytes cultured in medium with bovine serum. For both PHA and Con A, optimal incorporation of radioactivity was achieved when cells were pulsed with [3H-methyl]-thymidine 72 h after addition of mitogen. The medium based on TCM-199 + horse serum could support interleukin-2-induced proliferation. Furthermore, known inhibitors of lymphocyte proliferation in sheep were effective in inhibiting lymphocytes cultured in this medium.