Anti b220 apc

At day 4 post-stimulation, B cells were washed and stained with anti-B220 APC (BioLegend) and either anti-IgG3-FITC (BD-Pharmingen), anti-IgG2b-PE (BioLegend), anti-IgG2a-PE (BioLegend) or anti-IgG1-FITC (BioLegend). Activated B cells from AID-deficient mice were included as negative controls. Data were obtained on 3 × 10⁴ viable cells by using a Coulter XL apparatus (Beckman Coulter).

At day 4 post-stimulation, cells were washed and stained with anti-B220 APC (BioLegend) and either anti-IgG3-FITC (BD-Pharmingen), anti-IgG1-FITC (BioLegend), anti-IgG2b-PE (BioLegend), anti-IgG2a-PE (BioLegend) or anti-IgA-FITC (BD-Pharmingen). Data were obtained on 3 × 10⁴ viable cells by using a Coulter XL apparatus (Beckman Coulter).

Cells were suspended in MACS buffer and incubated with anti-CD16/32 mAb (BioLegend, 101310) for Fc receptor blocking. Cells were stained with the following fluorochrome-conjugated antibodies: PerCP/Cy5.5 anti-CD45 (BioLegend, 103131), APC anti-CD45 (BioLegend, 103111), PE/Cy7 anti-CD11b (Biolegend, 101215), FITC anti-CD3 (BD Biosciences, 553061), PE anti-CD4 (BD Biosciences, 553049), APC anti-CD8 (BioLegend, 100712), PerCP/Cy5.5 anti-CD11c (BioLegend, 117328), PE/Cy7 anti-MHC II (BioLegend, 107630), FITC anti-CD11b (BioLegend, 101206), V421 anti-CD64 (BioLegend, 139309), PE anti-Ly6G (BioLegend, 127607), APC anti-B220 (BioLegend, 103211), and PE anti-NK1.1 (BD Biosciences, 553165). Stained cells were analyzed by BD FACSVerse (BD bioscience). Data analysis was performed using FlowJo software (Treestar).

RBC-depleted spleen cell suspensions prepared from WT and TAX1BP1^−/−mouse were labeled with CD11b and CD90.2 microbeads (Miltenyl Biotec). Then the B cells were enriched by negative selection using autoMACSpro separator (Miltenyl Biotec) according to the manufacturer’s instructions. The collected cells were stained with the following antibodies: APC anti-B220, PE anti-Fas, PE anti-CD138 (Syndecan-1), FITC anti mouse IgM, FITC anti-GL7 antigen (BioLegend) and 7-AAD (BioLegend). Single-cell suspensions of bone marrow from WT and TAX1BP1^−/− mouse were stained with following antibodies: PE/Cy7 anti-TCR β chain, 7-AAD, PerCP-Cy5.5 anti-Gr-1, APC anti-B220, and FITC anti-mouse IgM (BioLegend). Single-cell suspensions of total splenocytes from WT and TAX1BP1^−/− mouse were stained with different combinations of the following antibodies: PE/Cy7 anti-TCR β chain, PerCP-Cy5.5 anti-Gr-1, 7-AAD, FITC anti-mouse IgM, APC anti-CD23, PE anti-CD21 (BioLegend), or PerCP-Cy5.5 anti-Gr-1, 7-AAD, PE/Cy7 anti-B220, PE anti-CD93, FITC anti-mouse IgM and APC-CD23 (BioLegend). WT DT40 cells, TAX1BP1^−/−/− cells and TAX1BP1^−/−/−/Tax1bp1 cells were stained with FITC conjugated anti chicken IgM (Bethyl). FACS Verse (BD) and SH800 (Sony Biotechnology Inc.) were used for flow cytometry, and results were analyzed with FlowJo software.

Spleen cells were prepared and stained extracellularly for APC‐anti‐B220 (BioLegend; RA3‐6B2) and PE‐anti‐CD5 (BD; 53–7.3) as described above. Intracellular staining was performed using the ADG Fix&Perm Kit (Dianova). After the initial staining cells were washed with PBS, then fixed with paraformaldehyde‐containing buffer A for 10 min at room temperature followed by washing with PBS. Antibodies for intracellular staining were dissolved in saponin‐containing buffer B and the staining was performed for 30 min at room temperature. Subsequently, cells were first washed with PBS, then with PBS containing 0.1% BSA (Carl Roth), 2 mM EDTA (Carl Roth) and 2 mM sodium azide (Sigma‐Aldrich). The following antibodies were applied for intracellular staining: AF488‐anti‐Akt (pS473) (BD; M89‐61), AF488‐anti‐Btk (pY551)/Ikt (pY511) (BD; 24a), AF488‐anti‐PLC‐γ2 (pY759) (BD; K86‐689.37), AF488‐anti‐S6 (pS240) (BD; N4‐41), AF488‐anti‐S6 (pS235/236) (BD; N7‐548), AF488‐anti‐ZAP70 (pY319)/Syk (Y352) (BD; 17A/P‐ZAP70), purified hamster anti‐mouse BCL‐2 (BD; 3F11) with secondary antibody anti‐Armenian Hamster‐AF488 (Jackson Immunoresearch). AF488‐anti‐Mouse IgG1κ isotype control (BD; MOPC‐21) and purified armenian hamster IgG1κ isotype control (BD) were used.

Staining was performed with hybridoma supernatant 2.4G2 Fc block (ATCC HB197) in FACS staining buffer at 4°C. The following antibodies were purchased from Biolegend: biotin-anti-CD11c (N418), Brilliant Violet 650 anti-CD11c (N418), PE-anti-CD11b (M1/70), PE-anti-CD3 (17A2), PE-anti-CD8 (53–6.7), PE-anti-Gr-1 (RB6-8C5), PE-anti-CD19 (eBio1D3), PE-anti-TER119 (TER-119), PE-anti-Thy1.1 (HIS51), PE-anti-NK1.1 (PK136), APC-anti-B220 (RA3-6B2), FITC anti-TLR9 (M9.D6), Brilliant Violet 421 anti-B220 (RA3-6B2), PerCP-eFluor710-anti-Siglec-H (eBio440c), PerCP-eFluor710-anti-c-Kit (2B8), APC-anti-M-CSFR (AFS98), PE/Cy7-anti-IL-7Rα (A7R34), FITC-anti-Sca-1 (D7). PE-anti-MHCII (NIMR-4) and FITC-anti-MHCII (NIMR-4) were purchased from eBioscience, and FITC-anti-IFNα (RMMA-1) was purchased from PBL Assay Science. APC-Cy7-Streptavidin was used as the secondary antibody (Biolegend). Flow data were analyzed with FlowJo (FLOWJO LLC) software.