Red blood lysis buffer

Mouse spleen and lymph nodes (mesenteric for the T-cell populations and inguinal for B-cell populations) were harvested and crushed to obtain a single-cell suspension. In the spleen, red blood cells were removed by a red blood lysis buffer (Invitrogen, Life Technologies, USA). A total of 1 × 10⁶ cells were transferred to 96-well plates and stimulated for 5 h with RPMI, 5% FCS, 1% penicillin/streptavidin, phorbol-12-myristate-13-acrylate (50 ng/ml), and ionomycin (1 µg/ml) at 37°C, 5% CO₂. Brefeldin A and monensin (1 mg/ml, BD Biosciences, USA) were also added 2 h after stimulation. Cells were labeled with surface markers (CD3-FITC, CD4-PerCP-Cy5.5, CD8a-PE-Cy7, CD25-Brilliant Vio510; CD19-PerCP vio770, CD9 PE-vio770, BD Biosciences and Miltenyi Biotec, France) with the Fc blocking antibody anti-CD16/CD32 (BD Biosciences). Cells were fixed and permeabilized with a Fixation/Permeabilization Solution Kit (BD Biosciences) to perform intracellular labeling (Fox-P3-PE, IL-4-APC, IFN-γ-APC-Cy7, BD Biosciences). Cells were analyzed by flow cytometry on a BD FACSCanto™ II Cell Analyzer (BD Biosciences). Data were acquired with Diva 8.0 software and analyzed with FlowJo Software v10 (TreeStar, Williamson Way, USA).

Livers were perfused through the right chamber of the heart using 10 mL of PBS and the portal vein was cut to limit perfusion circulation to the liver. Briefly, a catheter with a 27G needle attached to a 12 mL syringe containing PBS was inserted in the right heart atrium. For flow cytometry analysis, the left lateral lobe of the liver was collected and weighed. Single-cell suspension of liver tissue were achieved by sharp dissection by scalpel (Swann-Morton, #0501) followed by enzymatic digestion in HBSS (ThermoFisher, #14025050) containing 0.5 mg/mL of collagenase type IV (Sigma-Aldrich, #C4-22-1G) and 0.05 mg/mL of DNAse (Roche, #10104159,001) at 37ºC for 30 min. After digestion, samples were homogenized by passing 5–6 times through an 18G needle attached to 2 mL syringe, and then transferring it to a 70 μm cell strainer placed on a 50 mL Falcon tube. 15 mL of PBS containing 10% FBS and 5 mM EDTA was passed through the cell strainer and samples subsequently centrifuged at 400 G force for 5 min at 4ºC. Then, the pellet was resuspended in red blood lysis buffer (Invitrogen, #00–4333-57) for 3 min at room temperature and 0.6 mL of PBS containing 1% FBS and 5 mM EDTA was added to stop the reaction. Then samples were centrifuged at 400 G force for 5 min at 4ºC to proceed with the staining for flow cytometry.