Canine lymphocyte cultures in vitro: evaluation of peripheral blood lymphocyte response to mitogens

Lymphocytes from dog peripheral blood have been stimulated in vitro with 3 different mitogens (Con A, PHA and PWM). Culture medium was RPMI 1640 enriched with either autologous plasma, fetal calf serum of a newly described defined serum substitute. In such cultures the number of surviving and activated cells was measured by cytofluorometry and the proliferation was assessed by thymidine incorporation. In unstimulated cultures, up to 70% of all cells had disappeared (died) during the first 42 hours of incubation, whereas the number of viable cells was reduced to 50-60% in mitogen stimulated cultures. Of the surviving lymphocytes, between 25-40% of the cells appeared to have an elevated RNA-content (activated or G1 cells). By comparison between thymidine incorporation and number of mitogen induced G1 cells, a very high correlation was found (r=0.92). However, the Slope of the regression line was much lower than expected. The low thymidine incorporation per activated cell was primarily related to the high cell death and a resulting dilution of tritiated thymidine. Indeed, preliminary results suggested that the same thymidine incorporation per G1b cells could be obtained if peripheral blood lymphocytes were washed immediately before pulsing as could be obtained with lymph node cells without washing.