Anti f4 80

LNs and spleen tissues were prepared using mechanical dissociation of minced tissue to obtain single cell suspensions. The following antibodies were used for flow cytometry: anti-Ly6G/Ly6C (Cat #58-5931-82), anti-Cd11b (Cat #12-0112-82), anti-CD86 (Cat #11-0862-82), anti-F4/80 (Cat #17-4801-82), anti-MHCII (Cat #13-5321-82), and anti-CD11c (Cat #11-0116-42) all from eBioscience, San Diego, CA) and anti-CD206 (clone MR5D3; BioRad). After being stained with a standard protocol, all events were analyzed with FlowJo software (FlowJo, LLC, Ashland, OR) and frequencies among live cells were obtained.

Spleens were homogenized by manual disruption and red cells were lysed as described above. The cells were resuspended at 4°C in FACS buffer (PBS, 5% FBS and 0.1% sodium azide). Antibodies were incubated with cells for 30 min at 4°C at the indicated dilution and washed with FACS buffer before resuspension in fresh FACS buffer. Samples were analyzed on a Cyan flow cytometer (Dako) or an LSR-II flow cytometer (BD). The antibodies used were as follows: anti-Ly6G (48–5931-82; eBioscience; dilution 1/200), anti-CD11c (17–0114-81; eBioscience; dilution 1/200), anti-CD19 (48–0193-82; eBioscience; 1/200), anti CD11b (17–0112-82 or 45–0112-80; eBioscience), anti-CD8a (11–0081-82; eBioscience; 1/400 dilution), anti-CD4 (558107; BD), anti-F4/80 (12–4801-82; eBioscience; 1/200), anti-CD115 (12–1152-82; eBioscience; 1/250), anti CD45.1 (25–0453-82; eBioscience; 1/200), anti-CD45.2 (17–0454-81; eBioscience; 1/200), and anti-Ly6C (53–5932; eBioscience; 1/200).

Cells were incubated with antibodies 4 °C 20 min. Cells were examined by Aria II Flow Cytometer (BD Bioscience, USA). Cells were gated as follows: DCs (F4/80⁻ CD11c⁺ IA/IE⁺), macrophages (CD11b⁺ F4/80⁺) and M1 macrophages (CD11b⁺ F4/80⁺ CD206⁻ MHC II^high), M2 macrophages (CD11b⁺ F4/80⁺ CD206⁺ MHC II^low). Intracellular cytokine staining: 2 µL mL⁻¹ Cell Activation Cocktail (with Brefeldin A) (Cat: 423,303, Biolegend, USA) was used to incubate cells at 37 °C in a CO₂ incubator for 6 h. Then, the Fixation/Permeabilization Solution Kit (Cat: 554,714, BD Biosciences, USA) was used to stimulus cells. After cell fixation and permeabilization (fixation/permeabilization solution, 100 uL/10⁶ cells, 4 °C, 30 min), the BD Perm/Wash™ Buffer is used to wash the cells and to dilute the IFN-γ antibody for staining (4 °C, 30 min). Antibodies: The following were purchased from BioLegend: anti-IA/IE (1:3200, Clone: M5/114.15.2, Cat: 107,630), anti-CD206 (1:200, Clone: C068C2, Cat: 141,705), anti-Ki67 (1:200, Clone: 16A8, Cat: 652,403), anti-IFN-γ (1:100, Clone: XMG1.2, Cat: 505,805). The following were purchased from eBioscience: anti-CD11b (1:200, Clone: M1/70, Cat: 47–0112-82), anti-CD11c (1:200, Clone: N418, Cat: 45–0114-82), anti-F4/80 (1:200, Clone: BM8, Cat: 17–4801-82).

Hepatic and adipose immune cells were pre‐incubated with anti‐mouse CD16/32 Fc blocker (BD Pharmingen, USA), followed by staining with the Live/Dead marker anti‐FVD‐APC‐Cy7 (all supplied by eBioscience, San Diego, CA, USA). The fluorochrome‐conjugated antibodies used in this study were anti‐CD45, anti‐CD3, anti‐NK1.1, anti‐CD4, anti‐CD8, anti‐CD44, anti‐CD62L, anti‐CD11b, anti‐F4/80, anti‐Ly6C, anti‐Ly6C, and anti‐Siglec‐F (all supplied by eBioscience, San Diego, CA, USA). Liver mononuclear cells were stimulated with phorbol‐myristate acetate/ionomycin/brefeldin A/monensin for 5 hr in vitro. The cells were fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA). The permeabilized cells were washed with FACS buffer and resuspended in 1% formaldehyde and stained for intracellular cytokines with anti‐IFN‐γ‐PE‐Cy7, anti‐TNF‐α‐APC, and anti‐IL‐17A‐APC fluorochrome‐conjugated antibodies. Stained cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

To purify KCs, Ly6C^hi and Ly6C^low IMs, liver NPCs were incubated with normal rat serum (Sigma) and anti-mouse FcγRII/III (Becton Dickinson, Franklin Lakes, NJ, USA) to minimize nonspecific antibody binding. Subsequently, the cells were stained with anti-CD45, anti-Ly6C, anti-Ly6G, anti-CD19, anti-SiglecF (Becton Dickinson) and anti-F4/80, anti-CD11b, anti-NK1.1 and anti-CD3 (eBioscience, San Diego, CA, USA), and sorted using a BD FACSAria II Cell Sorter (BD Bioscience, San Jose, CA, USA).