Fig. (3) IFN-γ production in mixed and transwell cultures of DO11.10 CD4+ T cells and elicited peritoneal macrophages. T cells and macrophages were co-cultured or cultured in transwell. Peritoneal macrophages were isolated and cultured in the presence of medium alone or viable Salmonella. Following exposure for 45 minutes, cells were washed to remove any extracellular bacteria with gentamicin-containing medium. Magnetically separated splenic DO11.10 CD4 + T cells, B cells, and OVA peptide were co-cultured or separated by transwells (A). To evaluate the role of IL-12, some mixed cultures were supplemented with anti-IL-12p40 antibody or isotype control antibody (B). Levels of IFN-γ in 48-hour supernatants were measured by enzyme immunoassay, and are reported as mean values of triplicate determinations. These studies were performed three times with similar results.