Cd19 bv421

Surface staining was performed in ice-cold PBS with 2% FCS for 15 min in the presence of FcγRII/III blocking antibody (CD16/32). The cells were surface-stained with the relevant antibodies in PBS + 2% FBS for an additional 20 min at 4 °C.
The following fluorochrome-conjugated monoclonal antibodies were used for cell phenotyping: B220 APC (clone RA3-6B2), CD11b PE (clone M1-70), CD138 PE (clone 281-2), CD19 FITC (clone 6D5), CD19 BV421 (clone C068c2), CD206 PE-Cy7 (clone C068c2), CD4 PE (clone RM4-5), CD5 PE-Cy7 (clone 53–7.3), CD8a Ly-2 APC/Fire 750 (clone 53–6.7), F4/80 APC-Cy7 (clone BM8), F4/80 APC (clone BM9), IA-d Alexa Fluor 647 (clone 39–10-8), SIRPα APC (clone 15–414) from Biolegend and CD3 eFlour 660 (clone 17A2). Viability dye eFluor 780 reagent (eBioscience, USA) or propidium iodide were used for live/dead cell discrimination.
Flow cytometry was conducted on a FACSCanto II flow cytometer or FACSAria III Cell Sorter (BD Biosciences), and data analysis was performed using the FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Heparinized whole blood was used for flow cytometry using the following antibody cocktail: CD45-PerCP (Clone 30-F11; Biolegend 103130), CD3-eFLUO450 (Clone 17A2; eBioscience 48-0032), NK1.1-PE (Clone PK136; BD557391), Ly6G-APC-CY7 (Clone 1A8; BD560600), CD11b PE-CY7 (Clone M1/70; BD552850), Ly6C-APC (Clone 1G7.G10; Miltenyi 130-093-136), CD19-APC-H7 (Clone 1D3, eBioscience 47-0193) and Siglec-F-PE (Clone E50-2440; BD552126). Further, cells from the whole brain were isolated for FACS. Anesthetized mice were perfused with PBS to remove the peripheral blood. Subsequently, brains were dissected, mechanically dissociated and digested with a collagenase mix (including collagenase IX, collagenase I, DNAse and RPMI-HEPES). Immune cells were separated using a Percoll-gradient and stained with CD45 PerCP (Clone 30-F11; Biolegend 103130), CD11c PE-Cy7 (Clone N418, eBioscience 25-0114)), F4/80 (Clone BM8; Biolegend 123116), CD11b BV510 (Clone M1/70; Biolegend 101245), CD3 BV421 (Clone 17A2; eBioscience 48-0032-82), CD19 BV421 (Clone 6D5; Biolegend 115538), Ly6G BV421 (Clone eBio927; eBioscience 48-3172), MHC II-APC-eFluor780 (Clone M5/114.15.2; eBioscience 47-5321-82). All samples were measured with a FACS-Canto II (BD Biosciences). Results were analysed with FACSdiva version 8 (BD Biosciences).

Immunophenotyping of lymphocytes was performed by multicolor flow cytometry. Heparinized whole blood samples were immediately stained for 15 minutes with the following antibodies: CD3-APC/Cy7 (UCHT, BioLegend; San Diego, CA, USA), CD4-HorizonV500 (RPA-T4, BD Biosciences; San Jose, CA, USA), CD45RA-BV421 (HI-100, BioLegend), CXCR5-PerCP/Cy5.5 (J252D4, BioLegend), CXCR3-APC (1C6, BD Biosciences), CCR6-PE (11A9, BD Biosciences), CCR7-PE/Cy7 (G043H7, BioLegend), PD-1-FITC (EH12.2H7, BioLegend), CD19-BV421 (HIB19, BioLegend), CD20-APC (2H7, BioLegend), CD27-APC/Cy7 (O323, BioLegend), and CD38-FITC (HIT2, BioLegend). Circulating Tfh cells were defined as CD3⁺CD4⁺CD45RA^-CXCR5⁺ cells, and Tfh1, Tfh2, or Tfh17 cell subsets as CXCR3⁺CCR6^- cells, CXCR3^-CCR6^- cells, or CXCR3^-CCR6⁺ cells among Tfh cells, respectively [19 (link), 20 (link)]. Activated Tfh cells were defined as the CCR7^lowPD-1^high cells among Tfh cells [26 (link)]. CD19⁺CD20^-CD27⁺CD38⁺ cells were defined as plasmablasts. Red blood cells were lysed with FACS Lysing Solution (BD Biosciences). All samples were analyzed with a FACS Aria III (BD Biosciences), and data were analyzed with FlowJo v.7.6.4 Software (Tree Star, Stanford University, CA, USA). Proportions of lymphocyte subsets were determined by the combination of surface marker staining, with exclusion of doublets by forward and side scatter.

Multicolour flow cytometry measurements were performed and analysed as described previously^{27 (link)}. Briefly, isolated SVF cells, which were obtained following collagenase digestion and lysis of red blood cells, were stained for flow cytometry using two antibody cocktails. Cocktail 1 included CD45-PE-Cy7 (BD 557748), CD3-fitc (BD 561807), CD19-fitc (BD 555412), CD56-fitc (BD 562794), CD66b-fitc (BD 555724), CD11b-BV421 (Biologend 301324), and CD11c-APC-Cy7 (Biolegend 337218). Cocktail 2 included CD45-PE-Cy7 (BD 557748), CD3-V500 (BD 561416), CD4-PerCP (Biolegend 300528), CD8 APC-H7 (BD 641400), CD19-BV421 (Biolegend 302234), and CD56-APC (Biolegend 318310). Samples were measured with a FACS-Canto II (BD Biosciences). Results were analysed with FACSdiva (BD Biosciences) and FlowJo software. Since weight of the AT samples was unavailable, data are expressed as percentage of cells relative to total number of cells (based on forward and side scatter plot).

Flow cytometric analysis was performed on BD® LSR II Flow Cytometer (Marshallscientific, USA), and data were analyzed with FlowJo10.0 software (Treestar, Ashland, OR, USA). Anti- mouse B220-FITC (103205, Biolegend, USA), CD43-PE-Cy7 (143210, Biolegend, USA), CD24-PE (101807, Biolegend, USA), CD19-PE-Cy7 (552854, BD, USA), CD19-BV421 (115520, Biolegend, USA), CD19-PE (115508, Biolegend, USA), CD69-FITC (104506, Biolegend, USA), CD5-PE (100608, Biolegend, USA), CD86-APC (105011, Biolegend, USA), CD95-PE (152608, Biolegend, USA), BP-1-Alexa Fluor 647 (108312, Biolegend, USA), IgD-APC (405714, Biolegend, USA), IgM-PE (406507, Biolegend, USA), and corresponding isotype control antibodies were purchased from Biolegend. Antibodies were listed in Supplementary Table 3.