In vitro stimulation of ovine lymphocytes by various mitogens

A technique for the separation and in vitro culture of ovine lymphocytes is described and applied to the study of their blastogenic responses to various mitogens. Lymphocytes were separated on a Ficoll-Triosil gradient, resuspended in RPMI 1640 medium supplemented with 10 per cent serum to a concentration of 1 x 10(6) cells/ml and cultured in 200 microliter volumes in microculture plates in the presence of mitogens for varying lengths of time. A total culture period of 66 h was found to be satisfactory with 1 muCi of [3H]-thymidine being added to each well 18 h before termination of culture. Optimal blastogenic stimulation of the lymphocytes occurred with phytohaemagglutinin at 2-5 microliter/ml, pokeweed mitogen at 10 microliter/ml, concanavalin A at 6-25 microgram/ml and lipopolysaccharide of Escherichia coli (LPS) at 6-25 microgram/ml. Proliferation due to stimulation by LPS appeared to be of a lower order than that achieved with the other mitogens tested.