The effects of cell density, incubation temperature, syngeneic serum, and syngeneic red blood cells on mouse lymphocyte responses in vitro.

Various parameters affecting the quantitative assessment of mouse mixed lymphocyte response (MLR) in microplates were investigated. In agreement with the findings of others cell density was found to be particularly critical, maximal proliferative efficiency occurring at 3 X 10(6) viable cells/cm2. Slight reduction in the degree of viable cell--cell interaction caused by either reduction in cell density or insertion of irradiated cells into the cultures greatly reduced the efficiency of the proliferation. An incubation temperature of 33-34 degrees C was found to have distinct advantages over 37 degrees C, there being improved cell survival and reproducibility at this lower temperature. Transformation of mouse lymphocytes was strongly inhibited by low concentrations of normal syngeneic serum, the degree of inhibition depending on the concentration of foetal calf serum (FCS) in culture and on the method of preparation of serum. Inhibition of mouse cell responses was also caused by syngeneic red blood cells (RBC), in contrast to the pronounced enhancement of guinea pig lymphocyte responses to phytohaemagglutinin (PHA) in the presence of syngeneic RBC.