**2.11 Obtaining of mononuclear cells to flow cytometry**

In the determined euthanasia points, the blood of each animal was collected by cardiac puncture using citrated saline (0.87% NaCl, 3.8% Na3C6H5O7) and diluted in the same volume of complete RPMI medium—RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO), supplemented with 10% fetal bovine serum (FBS) (CultiLab, Campinas, Brazil); 200 mM L-glutamine; 100 U/mL penicillin; and 10 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO). Cells were then added to a Ficoll–Hypaque (Histopaque 1077; Sigma-Aldrich) sedimentation gradient. After centrifugation at 1030 x g for 20 min at 21 C, without brake, the mononuclear cell (MCs) ring was collected. A lobe of the liver, mesenteric lymph nodes, and spleen were macerated in 4 mL of complete RPMI medium until complete disruption using a glass tissue homogenizer (Corning E.U.A.), being kept on ice throughout the process. Cardiac cells were obtained after organ perfusion with PBS (pH 7.2), subsequently cut into small fragments, and subjected to four cycles of dissociation using 0.2% type II collagenase in RPMI medium without FBS at 37°C, stirring for 30 min. The supernatant obtained after each dissociation cycle was collected in a single tube and kept on ice. Cells from blood, liver, spleen, mesenteric lymph nodes, and heart were washed twice (with centrifugation at 720 x g, 4°C, 5 min) in PBS-BSA-(Bovine Serum Albumin) with 10% FBS and resuspended with 3 mL of ACK (Ammonium-Chloride-Potassium) Lysing Buffer, to lyse of red blood cells, incubated for 5 min at room temperature and washed again. After that, cells were incubated in PBS-BSA with 10% horse serum (HS) for 30 min, washed again, resuspended with complete RPMI medium, and adjusted to 1x106 cells/well.
