Anti cd16 cd32 antibody

Staining was performed in PBS/2%FCS with anti-CD16/CD32 antibodies (BioLegend, Fell, Germany) to block FcR-binding and DAPI (Sigma-Aldrich, Munich, Germany) or Live/Dead (Thermo Fisher, Schwerte, Germany) to exclude dead cells. FACS was performed on a LSR II® (BD Biosciences) and data analyzed with FlowJo® (Treestar Inc., Ashland, OR). FACS-analysis/sorting antibodies are listed in Table S1.

Single-cell suspensions from spleens and the circulation obtained during the sacrifice were washed with PBS + 2% FBS and blocked with anti-CD16/CD32 antibodies (Biolegend, San Diego, CA, USA). Cells were stained with anti-F4/80, anti-CD11b, anti-Ly6C, and anti-Ly6G antibodies, or with anti-CD4, anti-CD8 and anti-CD25 antibodies (BioLeg-end, San Diego, CA, USA) for 30 min at 4 °C. Cells were washed twice and analyzed by flow cytometry using an Attune NxT (ThermoFisher^®) cytometer. For intracellular staining, cells were washed and incubated in FOXP3 Fix/Perm Buffer Set (BioLegend, San Diego, CA, USA) for 30 min following the manufacturer’s instructions and then stained with anti-Foxp3 antibody (Becton Dickinson, San Jose, CA; USA). Then, 5000 gated events were captured and analyzed. Data were analyzed using the FlowJo software V X (Tree Star).

Cells from two animals (IL17-eGFP mice for meninges and brain and C57BL/6 for NALT analysis) were combined and resuspended in 50 μl of FACS buffer (PBS, 2% FBS, 0.05% NaN₃) and incubated with 5 ng/μl of anti-CD16/CD32 antibodies (BioLegend, clone 93) for 10 minutes at 4°C to block nonspecific binding. Then, cells were stained for 15 minutes at 4°C with the following antibodies: CD45 (clone 30F.11, 2.5 ng/μl) CD11b (clone M1/70, 0.6 ng/μl), TCRβ (clone H57-597, 4 ng/μl), CD4 (clone RM4-5, 0.5 ng/μl), CD8 (clone 53–6.7, 2.5 ng/μL), CD19 (clone 6D5, 0.6 ng/μl), TCRγδ (clone GL3, 4 ng/μl) from BioLegend. Cells were washed with FACS buffer, resuspended in 200 μl of FACS buffer and samples were acquired on a MACSQuant10 cytometer (Miltenyi Biotec). Acquired data were analyzed using FlowJo software (Version 10, Tree Star).

Single-cell suspensions obtained from the blood, spleen and kidney were stimulated at 37°C for 6 h at 5% CO2 in the presence of 50 ng/ml phorbol myristate acetate (Sigma-Aldrich), 1 μg/ml ionomycin (Sigma-Aldrich), and Brefeldin A Solution (BD Bioscience, USA). After incubation, the cells were blocked with anti-CD16/CD32 antibodies (Biolegend, USA) and then stained with the indicated antibodies for 20 min at 4°C. After fixation and permeabilization with Perm/Wash solution (BD Pharmingen, USA), the cells were stained with allophycocyanin-conjugated anti-IL22 monoclonal antibodies (Biolegend, USA) in the dark at 4°C for 30 min. Then, the cells were washed with Perm/Wash solution once and resuspended. Isotype controls were utilized to enable accurate compensation and to confirm the antibody specificity. The stained cells were analyzed by FCM using ACEA NovoCyte™ cytometer (ACEA Biosciences, USA) and analyzed with NovoExpress software (ACEA Biosciences, USA) and FlowJo software (Tree Star Inc, Ashland, OR, USA). Refer to the antibody table (Supplemental Table 2) for a complete list of antibodies.

Mouse spleens were grinded and connective tissues were removed to obtain splenocytes. Pelleted cells were resuspended by 2 ml ACK lysis buffer and incubated for 1 min to lyse red blood cells, followed by a wash with 10 ml 0.2% BSA (bovine serum albumin) in PBS. Pelleted splenocytes were resuspended in 0.2% BSA in PBS containing anti-CD16/CD32 antibodies (Biolegend) to block the Fc receptors before staining. After incubation for 10 min at 4°C, 50 μl diluted antibodies including anti-CD19, CD3, CD4, CD8, and NK1.1 antibody (Biolegend) were added and incubated for 30 min in the dark. Stained cells were washed twice with 0.2% BSA in PBS, resuspended in 300 μl 0.2% BSA in PBS, and then analyzed by CytoFLEX flow cytometer (Beckman Coulter). For intracellular IFN-γ staining, mice were injected with 20 mg/kg LPS and after 2.5 hr splenocytes were isolated. Splenocytes were fixed and permeabilized with 100 μl BD cytofix/cytoperm (BD biosciences) for 20 min after surface staining. BD Perm/Wash Buffer (BD biosciences) was used to wash the cells. Pelleted cells were resuspended in BD Perm/Wash Buffer containing anti-IFN-γ antibodies (Biolegend) for 30 min and analyzed by CytoFLEX flow cytometer.

For co-culture, flow cytometric analysis was performed using a BD Cytofix/Cytoperm Plus fixation/permeabilization protocol (BD Biosciences, San Jose, CA, USA), as previously described [52 (link)]. The co-cultured cells were blocked for nonspecific binding using anti-CD16/CD32 antibodies (BioLegend, San Diego, CA, USA, 101301) for 20 min, and the co-cultured cells were fixed and permeabilized with BD Cytofix/Cytoperm solution for 20 min at 4 °C. The co-cultured cells were washed with a BD Perm/Wash buffer, resuspended in a BD Perm/Wash buffer, and incubated with anti-CD206-PE (eBioscience, 12-2069), anti-ARG1-PE (BioLegend, 369704), anti-IL-10-eFluor 660 (eBioscience, 50-7108), anti-IFN-γ-PE (eBioscience, 12-7319), anti-TNF-α-APC (eBioscience, 17-7349), or anti-PD-L1-APC (BioLegend, 329707) for 30 min at 4 °C. The co-cultured cells were subsequently washed with a Perm/Wash buffer and resuspended in a Flow Cytometry Staining Buffer.