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.