The spleens of littermate or TCR transgenic mice were processed into a single cell suspension, as described previously (35 (link)). Splenocytes (1×105 cells/well) were seeded in a 96-well flat-bottomed tissue culture plate in complete media with 5 μg/ml C. muridarum AR Nigg, plasmid-deficient CM 3.1, C. trachomatis D/UW-3/Cx, or recombinant ovalbumin (Sigma) for 6 days. Splenocytes were treated with 20 U/mL murine IL-2 (Peprotech) over the final 48 hours. Cells were treated with 20 μl of Alamar Blue (Biosource) 6 hours before the end of the 6-day culture, and proliferation was measured at 530-nm excitation/590-nm emission with a Biotek fluorescence microplate reader.
Alternatively, transgenic or polyclonal CD4+ T cells were isolated from the spleens of naïve TCR transgenic or wild-type C57BL/6J mice by negative magnetic selection (EasySep™ Mouse CD4 T cell). Isolated CD4+ T cells were co-cultured at a 1:5 ratio with bone marrow-derived dendritic cells (BMDCs) for 3 days in the presence or absence of C. muridarum AR Nigg (5μg/mL). Expression of CD69 and Ki67 was determined by FACS surface and nuclear staining respectively, as described previously (36 (link)). Supernatants from dendritic-cell stimulated CD4+ T cells were collected and IL-2 concentrations determined by ELISA.
Alternatively, transgenic or polyclonal CD4+ T cells were isolated from the spleens of naïve TCR transgenic or wild-type C57BL/6J mice by negative magnetic selection (EasySep™ Mouse CD4 T cell). Isolated CD4+ T cells were co-cultured at a 1:5 ratio with bone marrow-derived dendritic cells (BMDCs) for 3 days in the presence or absence of C. muridarum AR Nigg (5μg/mL). Expression of CD69 and Ki67 was determined by FACS surface and nuclear staining respectively, as described previously (36 (link)). Supernatants from dendritic-cell stimulated CD4+ T cells were collected and IL-2 concentrations determined by ELISA.