CD8+ T cell purification and co-culture with stimulated macrophages
CD8+ T cells were purified by magnetic negative selection using a CD4+ T cells isolation kit (Miltenyi Biotec, Bergish Gladbach, Germany) from spleen cells according to the manufacturer’s instructions. Briefly, spleens were removed from euthanized mice, and mechanically perfused by injection of cold PBS. Splenocytes were rinsed with supplemented DMEM pH 7.4, and pelleted by centrifugation for 10 min at 600 x g, 18ºC36. Cells were resuspended in lysis solution (Tris base 0.17 M pH 7.2 and NH4Cl 0.16 M) for 15 minutes at 37º C. Consecutively, cells were completed with PBS and pelleted by centrifugation at the same conditions mentioned. Supernatant was decanted, and the splenocyte pellet was resuspended in supplemented DMEM, cell viability was assessed by trypan blue exclusion and cell numbers were determined by Neubauer chamber. A total of 2x105 of purified CD8+ T cells by magnetic negative selection were co-cultured with peritoneal murine macrophages previously stimulated with 100, 50, 25 or 12.5 µg/ml of recombinant TSA-1-C4 plus Tc24-C4 antigen combination at 1:2 proportion (lymphocyte:macrophages). Supernatants from peritoneal macrophages stimulated with recombinant proteins were removed through DMEM washing, hence, co-culture was performed using a 24-well plate and incubated under 5% CO2 and 37 °C for 48 h. Later, supernatants were collected to measure cytokine production. Macrophages stimulated with LPS (1 µg/ml) or MTX (10 µg/ml) were used as positive controls and non-stimulated macrophages (only supplemented DMEM) were used as basal control.