Isolation, culture and stimulation of murine macrophages
Mice were euthanized by phenol-chloroform and sacrificed by cervical dislocation. In a laminar flow hood, the abdomen was disinfected with ethanol 70%, and resident macrophages were harvested by injection of cold phosphate-buffered saline (PBS) into the peritoneal cavity, consecutively, were poured in sterile tubes. Cells were pelleted by centrifugation for 10 min at 600 x g and 4°C, consecutively, cells were resuspended in Dulbecco´s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco), in an atmosphere of 5% CO2 and 95% humidity at 37 ºC35,36. Cell viability was assessed by trypan blue exclusion and cell quantification was determined by Neubauer chamber. In order to obtain adherent cells (macrophages), we added 1x105 cells in a 24-well plate under 5% CO2 and 37 °C for 48 h, afterwards, non-adherent cells were removed by DMEM washing. In order to stimulate cells, we added recombinant TSA-1-C4 and Tc24-C4 antigens mixed in equal proportion to reach final concentrations per well to 100, 50, 25, 12.5, and 6 µg/ml. Stimulated cells remained on incubation for 48 h. at same conditions mentioned previously. The maximum concentration tested here was based on the previous reports that used recombinant proteins for immunization assays31. Individual stimulation with recombinant TSA-1-C4 or Tc24-C4 antigens were also included.