Rat mixed lymphocyte culture: optimization of culture conditions

We have systematically analysed the various parameters of rat mixed lymphocyte culture (MLC), aiming at defining optimal conditions in analytical (micro) MLC and at the production of maximal numbers of blast cells in preparative (maxi) MLC. Treatment of both responder and stimulator cells, or at least the responder cells, with N-acyl-neuraminidase allowed a good and reproducible analytical MLC response. Responses with a maximal resolution between the stimulated versus nonstimulated control cultures were obtained in the presence of rat sera, BN serum being superior to Lewis and AO serum in supporting the response. Rat sera derived from DA and HO strains, fetal calf serum, and human serum were not good. Spleen cells, lymph node cells, and density-separated blood leucocytes were good responders, and spleen cells were good stimulators, provided the spleen cells were prepurified from most of the phagocytic cells with iron powder plus magnet. Similar culture conditions were applicable also for the maxi-MLC assay. The number of blast cells generated from a single spleen could, however, be increased by a factor of 10, if the responder cell donor was primed intravenously with 10 x 10(6) stimulator-strain spleen cells 72 h before being killed. The responses both in the non-primed and in the primed cultures were specific, since the background stimulation was negligible and the cytotoxic effect by the primed cells in the cell-mediated lysis assay was immunologically specific.