Analysis of the in vitro activation and proliferation process in lymphocytes deriving from canine thymus and mesenteric lymphnode

Lymphocytes from dog thymus and mesenteric lymphnodes have been stimulated in vitro with 3 different mitogens. Culture medium was enriched with either autologous plasma, fetal calf serum or a newly described defined serum substitute. In such cultures the number of surviving and activated cells was quantified by cytofluorometry and the proliferation was assessed by thymidine incorporation. Results obtained with the 3 media were very similar. A significant number of lymphocytes died during the first 42 hours of incubation. There was a tendency toward more surviving cells with fetal calf serum and the serum substitute, whereas more activated (G₁) cells and higher thymidine incorporation could be observed with autologous plasma. Furthermore, when results obtained with various mitogen concentrations and from individual dogs were analyzed, a high correlation between “highly activated (G_1b) cells and thymidine incorporation was found, i.e. r=0.84 for thymocytes and 0.68 for lymphnode cells. The correlation between all G₁ (G_1a+b) cells and thymidine incorporation was lower or absent (r=0.02 and 0.55, respectively). It is concluded from these results that the population of G_1b cells have received all required signals necessary for proliferation whereas the total G₁ cell population also include activated cells, which are not obligatorily undergoing subsequent proliferation