Materials and Methods

Animals employed as cell donors were rats of the isogenic L, DA, and BN strains and their Fl hybrids. These strains are known to differ by alleles at the important AgB histocompatibility locus (17, 18).

Cultures were prepared from peripheral blood leucocyte suspensions obtained by dextran sedimentation from whole blood collected by cardiac puncture. Procedures for the preparation and culture of the leucocyte suspensions have been described in detail elsewhere (11). Each culture consisted of a mixture of 2 million lymphocytes--1 million each from a parental strain and an FI donor. The use of this experimental design provides a unidirectional response since previous studies have shown that proliferating ceils in parental F1 cultures are almost exclusively of parental origin (11, 12, 14). Control, unmixed cultures, consisted of 2 million lymphocytes from parental or F1 donors. All cultures were conducted in triplicate unless specified to the contrary, in a volume of 1 ml in 13 X 100 mm disposable flint glass tubes) They were maintained in a humid atmosphere of 5% CO,-95% air at 37 C. The medium s was supplemented with fresh BN serum (10%) prepared on the day of culturing from BN strain donors. The proliferative response of the MLI was assessed by one or more of three procedures: