This paper describes an in vitro system for studying the factors that control and regulate cellular events in the immune response. It was previously reported that dissociated spleen cell suspensions from normal mice could be immunized in vitro to heterologous erythrocytes. The response is primary, occurring after the first exposure to the erythrocyte antigen. The response was measured by the increase in hemolytic plaque-forming cells and antibody levels in the culture supernatants. Critical conditions for the culture and immunization included low oxygen tension, gentle agitation, fetal bovine serum, adequate spleen cell density, and daily feeding with a nutritional mixture. The paper presents a detailed account of the experimental system and compares in vitro and in vivo responses under various conditions. The in vitro response closely mirrors in vivo responses in terms of size, early kinetics, antigen dose, and passive antibody inhibition. However, the in vitro response shows greater discrimination between homologous erythrocyte antigens and does not show a limitation in the increase of 19S antibody-forming cells. The study describes the materials and methods used, including mice, biological reagents, chemical reagents, and cell preparation. The standard cell culture conditions, including the use of a specific medium and nutritional mixture, are detailed. The hemolytic plaque assay is described, along with the results of in vitro immunization with sheep erythrocytes and comparisons with other erythrocytes. The results show that in vitro immunization with sheep erythrocytes leads to a significant increase in plaque-forming cells. The response is influenced by oxygen tension, motion, spleen cell concentration, and fetal bovine serum. The study also examines the effect of passive antibody on the response and the continuation of the response beyond day 4. The discussion highlights the similarities between in vitro and in vivo responses, as well as the differences in antigen discrimination and response duration. The study concludes that the in vitro system is useful for studying immune responses and provides insights into the mechanisms regulating the immune response. The authors thank colleagues for their assistance and acknowledge the support of the National Institutes of Health.This paper describes an in vitro system for studying the factors that control and regulate cellular events in the immune response. It was previously reported that dissociated spleen cell suspensions from normal mice could be immunized in vitro to heterologous erythrocytes. The response is primary, occurring after the first exposure to the erythrocyte antigen. The response was measured by the increase in hemolytic plaque-forming cells and antibody levels in the culture supernatants. Critical conditions for the culture and immunization included low oxygen tension, gentle agitation, fetal bovine serum, adequate spleen cell density, and daily feeding with a nutritional mixture. The paper presents a detailed account of the experimental system and compares in vitro and in vivo responses under various conditions. The in vitro response closely mirrors in vivo responses in terms of size, early kinetics, antigen dose, and passive antibody inhibition. However, the in vitro response shows greater discrimination between homologous erythrocyte antigens and does not show a limitation in the increase of 19S antibody-forming cells. The study describes the materials and methods used, including mice, biological reagents, chemical reagents, and cell preparation. The standard cell culture conditions, including the use of a specific medium and nutritional mixture, are detailed. The hemolytic plaque assay is described, along with the results of in vitro immunization with sheep erythrocytes and comparisons with other erythrocytes. The results show that in vitro immunization with sheep erythrocytes leads to a significant increase in plaque-forming cells. The response is influenced by oxygen tension, motion, spleen cell concentration, and fetal bovine serum. The study also examines the effect of passive antibody on the response and the continuation of the response beyond day 4. The discussion highlights the similarities between in vitro and in vivo responses, as well as the differences in antigen discrimination and response duration. The study concludes that the in vitro system is useful for studying immune responses and provides insights into the mechanisms regulating the immune response. The authors thank colleagues for their assistance and acknowledge the support of the National Institutes of Health.