Facsdiva v8

Nonspecific binding was blocked with an anti-FcγIII/II antibody (anti-CD16/32; BD Pharmingen, Heidelberg, Germany). The following antibodies and conjugates were used in the experiments in appropriate combinations: anti-CD5-phycoerytrhin (PE)/Cy7 (clone 53-7.3) (Biolegend, San Diego, CA, USA), anti-CD19-eFluor 660 (clone 1D3) (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), anti-CD23-PE (clone B3B4) (eBioscience), anti-CD3-FITC (clone 145-2c11), anti-CD11b-APC-eFluor 780 (clone M1/70) (eBioscience), anti-IgD-V450 (clone 11-26c.2a) (BD Biosciences), and anti-IgM-BV650 (clone R6-60.2) (BD Biosciences). For dead cell exclusion, cells were stained with 7-AAD Viability Staining Solution (Biolegend) before analysis. Stained cells were analysed on a BD LSR II Flow Cytometer (BD Biosciences) and evaluated with FlowJo software (Version 10, LLC, Ashland, OR, USA). The applied gating strategy is shown in Supplemental Figure S2. Fluorescence-activated cell sorting was performed on a FACSAria III cell sorter and gated in FACSDiva v8.0 (BD Biosciences). B1a B cells were identified as CD19⁺CD5⁺CD23⁻IgM⁺IgD⁻, B1b B cells as CD19⁺CD5⁻CD23⁻IgM⁺IgD⁻, and B2 B cells as CD19⁺CD5⁻CD23⁺IgM^lowIgD⁺.

For surface antigen identification, cellular pellets obtained after CSF centrifugation were resuspended in the residual volume (about 100 µl), stained with the appropriate amounts of monoclonal antibodies for 30 minutes at 4°C in the dark, washed twice with PBS, and analyzed in a FACSCanto II flow cytometer (BD Biosciences). For intracellular cytokine detection, cellular pellets were stimulated and stained for flow cytometry analysis as described previously (20 (link)). In brief, cellular pellets were incubated for 4 hours at 37°C in 5% CO2 with 50 ng/ml Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) and 750 ng/ml Ionomycin (Sigma-Aldrich) in presence of 2 µg/ml Brefeldin A (GolgiPlug, BD Biosciences) and 2.1 µM Monensin (Golgi Stop, BD Biosciences). After incubation, cells were washed and stained with the monoclonal antibodies recognizing the surface antigens. Then, cells were washed, fixed and permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), washed twice and subjected to intracellular staining with monoclonal antibodies recognizing different cytokines. Then, cells were washed and analyzed in a FACSCanto II flow cytometer. Data analysis was performed using the software FACSDiva V.8.0 (BD Biosciences) and the gating strategy shown in Supplementary Figures 2, 3. All labeled cells were acquired to calculate total cell numbers.