Percoll solution

Non-epithelial cells were prepared from the lamina propria of the small intestine as described previously.^{71 (link)} After epithelium was removed by incubation in 2 mM EDTA, the remaining intestinal tissue was chopped into small pieces, and digested with collagenase (0.5 mg/ml, Wako). The obtained cell suspension was suspended in 40% Percoll solution (GE Healthcare) and loaded on a 80% Percoll solution. After centrifugation for 20 min at 880 × g at room temperature, cells at interface between the two Percoll solutions were harvested. After stimulation for 4 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of GolgiPlug (1:1000 dilution), cell surface was stained with FVD506 (eBioscience 65-0866-14), APC/Cy7-labeled anti-CD45.2 antibody (BioLegend 109824, clone 104), APC-labeled anti-CD4 antibody (TONBO 20-0042, clone RM4-5), PE/Cy7-labeled TCRβ antibody (TONBO 60-5961, clone H57-597). The cells were fixed with Intracellular Fixation Buffer (eBioscience 00-8222) and then perforated in Permeabilization Buffer (eBioscience 00-8333). Intracellular cytokines were stained with PE-labeled anti-IL-17A (BioLegend 506904, clone TC11-18H10) and FITC-labeled anti-IFN-γ (BioLegend 505806, clone XMG1.2) in Permeabilization Buffer, and analyzed using LSR Fortessa^TM X-20 Cell Analyzer (BD Bioscience) and FlowJo (BD Bioscience).

For tissue isolation, mice were killed and the livers were removed. Liver-resident and recruited macrophages were separated as described previously^{40 (link)}. Briefly, the livers were minced and filtered without any enzymatic digestion. The constituent cells were resuspended in 25% Percoll solution (GE Healthcare Life Science) and gradient-centrifuged with 50% Percoll solution. After blocking with an anti-FcR antibody (CD16/32; BD Pharmingen, San Diego, CA) for 30 minutes at 4 °C, the cells were incubated with specific fluorescence-labeled monoclonal antibodies at 4 °C for 30 minutes. For sorting of liver-resident and recruited macrophages, anti-F4/80 (eBioscience), anti-7-AAD (eBioscience), anti-CD45.2 (BD Pharmingen) and anti-CD11b (BD Pharmingen) monoclonal antibodies were used.

When staining cells from B16.F10 and LL/2 tumors, lymph nodes, frontal cortex, lungs or thymus, mice were euthanized, organs harvested and stored in T cell media on ice. Organs were cut into smaller pieces and incubated for 30 min with reagents from a tumor dissociation kit (Miltenyi Biotec, catalog: 130-096-730), strained through a 70 μm nylon mesh, washed and resuspended in 100% Percoll solution (GE Healthcare, catalog: 17-0891-01), and layered carefully on top of 3 mL of 80% and 40% Percoll solution. After 30 min at 2000g, cells at the 80–40% interphase were collected and stained.

Hepatic mononuclear cells were prepared as previously described [35 (link)]. Briefly, liver of mice was removed and washed with Ca and Mg free Dulbecco’s Phosphate Buffered Saline (DPBS). After pressing through a 200-gauge stainless steel mesh, the cell mixture was resuspended in 40% Percoll solution (General Electric Company, USA), followed by overlaying gently onto 70% Percoll solution. Then, this cell mixture was centrifuged at 1260×g for 30 min at room temperature. The interface cells between the Percoll solutions were aspirated and washed twice with PBS medium. Single cell suspensions were resuspended in cell staining solution (PBS with 2% FCS) for flow cytometry.

As we described previously in our study^{43 (link)}, the ear samples were minced on day 15 and enzymatically digested with 6.47 mL of RPMI 1640 medium-10% fetal bovine serum containing 2 mg/mL crude collagenase D (Roche, Basel, Switzerland), 1.5 mg/mL hyaluronidase (Sigma-Aldrich), and 0.03 mg/mL DNase I (Sigma-Aldrich) at 37 °C for 90 min to prepare the cell suspensions for flow cytometry. Digested cells were passed through a 70-μm cell Falcon Cell Strainer (BD Biosciences) to generate a single-cell suspension. The cell suspension was then centrifuged at 460 × g for 20 min. The pellet was resuspended in 70% Percoll solution (GE Healthcare, Uppsala, Sweden), and overlaid with a 37% Percoll solution followed by centrifugation at 500 × g for 20 min at room temperature. Cells were aspirated from the Percoll interface and passed through a 70-μm cell strainer. The cells were harvested by centrifugation and washed with PBS.