Anti cd11b pe cy7

Before cytokine staining, T cells underwent in vitro stimulation with PMA (40 ng/ml), Ionomycin (2 μM), Monensin (4 μM) in 1 ml RPMI 1640 (containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 10 mM HEPES) for 4 h. Cells were resuspended in PBS supplemented with 0.5% BSA and 1 mM EDTA (FACS buffer) (splenocytes and lymphoid cells: 1 × 10⁶/tube, all available CNS mononuclear cells from each sample/tube) and stained with the antibodies listed below at 4˚C for 30 min. The anti-mouse antibodies used for flow cytometric analysis were the following: anti–CD45–APC-Cy7, anti-CD4-APC, anti-IFNγ-BV421, anti-IL-17–PerCP Cy5.5, anti-IL-10–FITC, anti-Foxp3-PE, anti-CD11b-PE-Cy7, anti-Ly6G-BV421, anti-MHC II-FITC, anti-Ly6C-PerCP Cy5.5, anti-CCR2- BV510 (all antibodies were from eBiosciences or BioLegend). After staining, the cells were analyzed with FACSVerse flow cytometer. Flow cytometric data were viewed and analyzed by FlowJo v10.

Cells from lymph nodes (5x10⁵) or ear homogenates (1x10⁶) were washed with PBS at 400 g for 5 min at 4°C and blocked with Human FcX (BioLegend) for 15 min, followed by staining with the antibody cocktail for 30 min at 4°C. Cells were then washed with a cytometry buffer (PBS with 5% FBS) at 400 g for 5 min and 4°C), then fixed with 4% formaldehyde (Sigma) for 15 min at 4°C. Cells were washed and resuspended in the cytometry buffer and stored in the dark at 4°C until acquisition. The following antibodies were used: anti-PD-L1-APC, anti-CD10-APC-780 (human, eBioscience); anti-CD45-APCcy7, anti-CD11b-FITC, anti-CD11b-PE, anti-CD11b-PEcy7, anti-Ly6G-PerCP, anti-Ly6G-FITC and anti-PD-L1-APC (murine, eBioscience). Acquisition of events (lymph node, 100,000 events; ear, all cells) was performed on a BD FACSAria™. The gate strategy was performed based on the selection of cell size (FSC) and composition (SSC). After identifying the main population, a gate of FSC-A (area) and FSC-H (weight) was used, where cellular doublets were excluded. Gates for positive events were established through Fluorescence Minus One (FMO) control. The data analyzes were performed using the FlowJo software.

Stromal vascular fraction (SVF) cells were harvested from the inguinal AT, and to block Fc receptors, they were incubated with FC block (Cat # 156604, TruStain FcX PLUS, anti-mouse CD16/32, BioLegend, London, UK) for 10 minutes. For the macrophages panel, cells were stained with anti-CD86-APC-R700 (Cat # 565479, BD Biosciences, San Jose, USA), anti-F4/80-APC antibody (Cat # 123116, BioLegend), and anti-CD11b PE-Cy7 (Cat # 25-0112-81, eBioscience, San Diego, USA). For the eosinophils panels, anti-Siglec-F BV421 (Cat # 562681, BD Biosciences), anti-CD11b APC (Cat # 101212, BioLegend), anti-CD63 PE(Cat# 564222, BD Biosciences), and anti-CD193 (CCR3) Alexa 647 (Cat# 144508, BioLegend) were used. After staining, cells were washed with a washing buffer (2% FCS in PBS) and analyzed using a BD FACSLyric™ flow cytometer (BD Biosciences).

Cell suspensions were Fc-Receptor blocked and stained with the following antibodies (all from Biolegend unless indicated otherwise): (Myeloid panel) anti-Ly6G-FITC (1A8; 1.25 μg/ml), anti-CD64-PE (X54-5/7.1; 2 μg/ml), anti-CD11c-PerCP-Cy5.5 (N418; 1 μg/ml), anti-CD11b-PE-Cy7 (M1/70; 0.2 μg/ml), anti-Ly6C-AlexaFluor700 (HK1.4; 2 μg/ml), anti-MHCII-APC-Cy7 (M5/114.15.2; 0.067 μg/ml), anti-TREM-1-eFluor660 (TR3MBL1 from eBioscience; 0.2 μg/ml), anti-CD45-BrilliantViolett (30-F11; 1 μg/ml). (Lymphocyte panel) anti-CD8b-FITC (53-5.8; 0.8 μg/ml), anti-TCRab-PE, anti-GL7-PerCP-Cy5.5 (GL7; 1 μg/ml), anti-CD19-PE-Cy7 (6D5; 0.5 μg/ml), anti-CD11b-PacificBlue (M1/70; 0.25 μg/ml), anti-NK1.1-AlexaFluor700 (PK136; 2.5 μg/ml), anti-CD4-APC-Cy7 (RM4-5; 0.25 μg/ml), anti-BTLA-AlexaFluor647 (6A6; 2.5 μg/ml), anti-CD45-BrilliantViolett (30-F11; 1 μg/ml). As an isotype control for the anti-TREM-1-e-Fluor660 a mouse anti rat IgG2a-eFluor660 antibody (eBR2a; 0.2 μg/ml) from eBioscience was used. Dead cells were excluded using DAPI (Invitrogen) at a final concentration of 0.5 μg/ml. All samples were acquired on a LSRII SORP (BD Biosciences, San Diego, CA) and analyzed using the FlowJo Software (Tree Star, Ashland, OR).

Dissected mouse livers were minced with a scalpel on ice and incubated at 37 ˚C in RPMI-1640 media containing 0.05% collagenase/dispase (11097113001; Roche) and 0.01% trypsin inhibitor (T7659; Sigma). Cell suspension was filtered through a 70 μm cell strainer into PBS and residual red blood cells lysed using red cell lysis buffer (11814389001; Roche). Cell suspensions at a concentration of 1 × 10⁷ cells/ml were the incubated for 45 min at 4 ˚C in the presence of anti-CD11b PE-Cy7 (25-0112-82, eBioscience), anti-Gr1 PE (12-5931-82; eBioscience), anti-CD45 PE (12-0451-82; eBioscience), anti-CXCR2-APC (149305; Biolegend), and Mouse BD Fc Block (553141, BD Bioscience). FACS analysis was performed using a FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software version 7.2.5 (Tree Star, Ashland, OR).