An improved method for obtaining optimal mitogenic responses in peripheral blood lymphocytes has been devised by utilizing autologous or homologous rainbow trout plasma. The use of 10% plasma in culture results in up to a 60-fold increase in the proliferative potential of the peripheral blood lymphocyte response to lipopolysaccharide when compared to the more routinely used fetal bovine serum. Furthermore, it has been observed that lymphocytes which were unresponsive to in vitro mitogenic challenge when cultured in fetal bovine serum, responded well when cultured in the presence of trout plasma. Also in contrast to previous mitogen studies where maximal stimulation was reported to occur on Day 4-5 of culture, the stimulatory effects of lipopolysaccharide were greatest on Day 10 when plasma was employed. Together these data suggest that former conditions of lymphocyte cell culture, employing only fetal bovine serum, not only fail to provide the optimal conditions for cell growth, but in many cases the essential conditions. However, these requirements are met by supplementation with trout plasma, which seems to contain heat-stable factors responsible for the enhanced mitogenic responsiveness.
