Single-cell suspensions from Peyer’s patches of 30-31 weeks old mice were first incubated with anti-mouse CD16/32 (BioLegend) and then labeled with B220-FITC– (BioLegend), CD19-APC– (BioLegend), CD38-Alexa700– (Thermo Scientific), and CD95/Fas-PE– (BD-Biosciences) conjugated antibodies. Non-germinal center (CD38+ Fas−) and germinal center B cells (CD38− Fas+) were sorted on an Aria BD sorter. Genomic DNA was extracted and the 5′ portions of JH4 (Igh) and JK5 (Igk) introns were amplified by PCR using Phusion High-Fidelity DNA Polymerase (Thermo Scientific). The 800 bp JH4 and 700 bp JK5 PCR products were gel extracted, cloned into a pCR2.1 vector using the TOPO TA Cloning Kit (Invitrogen) and sequenced. Mutations were quantified over 510 bp downstream JH4 and 536 bp downstream JK5 gene segments. Primers used for SHM analysis (Rouaud et al., 2013 (link), Sander et al., 2015 (link)) are listed in Table S5 .
B220 fitc
Manufactured by BioLegend
Sourced in United States
B220-FITC is a fluorescent-labeled antibody that binds to the B220 (CD45R) antigen, which is expressed on the surface of B lymphocytes. This product is intended for use in flow cytometry applications to identify and quantify B cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BioLegend (4 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (3 mentions)
Cd95 fas pe, by BD (3 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (3 mentions)
Lsrfortessa, by BD (3 mentions)
Nk1.1 pe, by BioLegend (3 mentions)
Cd3 apc, by BioLegend (3 mentions)
Cd38 alexa700, by Thermo Fisher Scientific (2 mentions)
Facsdiva software, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Cd4 pe, by BioLegend (2 mentions)
Cd8 pe, by BioLegend (2 mentions)
Cd11b fitc, by BioLegend (2 mentions)
Cd45 apc cy7, by BioLegend (2 mentions)
Cd11c pe cy7, by BioLegend (2 mentions)
Cd11b apc, by BioLegend (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Siglecf pe, by BD (2 mentions)
Cd4 pe cy7, by BioLegend (2 mentions)
Mhcii fitc, by BioLegend (2 mentions)
26 protocols using b220 fitc
1
Quantifying Somatic Hypermutation in Murine B Cells
2
Multiparametric Immune Cell Profiling
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1 × 106 cells were Fc blocked (Biolegend101320) and stained for 1 hour, on ice, in the dark, as previously described.20 (link) The following antibodies were used, B220−FITC (BD553087), I Ab PerCP−Cy5.5 (Biolegend116416), Ly6C PE or PECy7 (Biolegend128008, 128071), HLA−DR PECy7 (eBioscience25−9956−41/Biolegend307606), CD11b BV510 (BD562950/Biolegend101263), Ly6G BV605 (BD563005/Biolegend127639), CD11c APC (BD550261), CD3 A700 (eBioscience56−0032−82/Biolegend100216), Live Blue Fluorescent reactive dye (LifeTech L34962), and CD45 BV650 (Biolegend103151).
3
Multiparameter B-Cell Immunophenotyping
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The following reagents were used for cell staining: B220-FITC (Biolegend, 103206), CD19-APC eFluor780 (eBioscience, 47-0193-82), CD21-PE (Biolegend, 123410), CD23-biotin (BD, 553137), Brilliant Violet 605 Streptavidin (Biolegend, 405229), and propidium iodide (Sigma, P4864-10ML). Cells were applied to flow cytometry using a BD LSRFortessa with FACSDiva software (BD) and analyzed using FlowJo 10.3 software (Tree Star). Follicular and marginal zone B cells (Fig. 1M) and activated lymphoblasts (Fig. 3G) were isolated using a BD Influx cell sorter. For follicular and marginal zone B-cell proportions, splenic single cell suspensions were first gated on live B220+ CD19+ lymphocytes, followed by gating on CD21/35 and CD23 (follicular B cells: CD21/35lo CD23hi; marginal zone B cells: CD21/35hi CD23lo). Cell division and proliferation indices of CTV-stained cells were determined using the cell proliferation module, and cell cycle stages were determined using the cell cycle module of FlowJo 10.3 software (Tree Star).
4
Multiparameter Flow Cytometry Analysis
5
B Cell Immunophenotyping and Cytokine Analysis
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B cells were washed once in cold PBS containing 0.1% BSA (FACS buffer) before blocking with anti-FcγRII/FcγRIII (2.4G2, BD Pharmingen, San Diego, CA). Stainings were performed on ice using conjugated mAbs (eBioscience, San Diego, CA, if not stated otherwise), diluted 1:300 in FACS buffer for surface marker and 1:200 for intracellular cytokine staining followed by incubation for 20 min. After washing with FACS buffer, cells were analysed on a FACS Canto II (BD) using FlowJo software (Tree star, Ashland, OR). The following Abs were used: B220-FITC (#11-0452-86), CD5-PE-Cy7 (#25-0051-81), CD1d-PE (#12-0011-81), CD138-APC (#142506, Biolegend), IgM-FITC (#11-5890-85), IgG1-PE (#12-4015-82), CD4-FITC (#11-0041-82), IL-2-APC (#17-7021-81), IFNγ-APC (#17-7311-82) and IL-10-PerCP (#45-7101-80). Abs against TNF−α−PE (#130-092-245) and IL-17-PE (#130-094-296) were from Miltenyi Biotec. For intracellular staining, the fixation and permeabilization kit (Plus Brefeldin A; eBioscience, Cat. no. 88-8823-88) was used according to manufacturer's recommendation.
6
Quantitative RT-PCR Analysis of Bone Marrow and B-Cells
7
Isolation and Analysis of DCs from Draining Lymph Nodes
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Draining lymph nodes from DT-depleted Karma mice were harvested into Click’s EHAA media (FUJIfilm) and minced with 22-gauge needles. Minced tissues were digested with 100 μg/ml collagenase D (Millipore Sigma Cat No. 11088882001) and 17 μg/ml DNAse (Worthington Biochemical Cat. No. LS002145) for 30 min at 37 °C. Following digestion, cells were filtered through a 100-micron screen and washed with 5 mM EDTA and 2.5% FBS in EHAA media to stop the digestion. Cells were washed with FACS buffer and stained with B220 FITC 1:200 (Biolegend Cat. No. 103206), CD11c APC Cy7 1:400 (Biolegend Cat. No. 117324), MHC Class II (I-A/I-E) BV421 1:1000 (Biolegend Cat. No. 107631), and XCR1 BV785 1:200 (Biolegend Cat. No. 148225) or XCR1 BV650 1:200 (Biolegend Cat. No. 148220) antibodies in 10% 24G2 (Fc Block) for 30 min at 4 °C. Cells were washed twice with FACS buffer and run on BD Canto II flow cytometer or Beckman Coulter Cytoflex LX flow cytometer.
8
Multiparametric Flow Cytometry Analysis of Immune Cell Subsets
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The fluorescent coupled antibodies used in this study included CD11b-Pacific Blue (Biolegend, #101224), Ly6C-PE (Invitrogen, #12-5932-82), Ly6G-PE/Cy7 (Biolegend, #127618), Aldh2-FITC (Novus, #NBP2-70151F), Socs3-APC (Biorbyt, # orb1000608), fixable viability dye eFluor 660 (Invitrogen, #65-0864-14), FerroOrange (Dojindo, #F374), CFSE (Biolegend, #423801), IL6-PE (Biolegend, # 504503), B220-FITC (Biolegend, # 103205), Ki67-APC (Biolegend, # 350514), SLC7A11 (Invitrogen, #MA5-44922, inhouse coupled to A647), DAPI (Invitrogen, #D1306), Lipid Peroxidation/LiperFluo kit (Invitrogen, #C10445), and FerroOrange Kit (Amerigo, #F374).
9
Flow Cytometric Enumeration of Eosinophils
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For detection of eosinophils, red blood cells were lysed using ammonium chloride lysis buffer (Sigma-Aldrich; Merck KGaA). Absolute counting beads (cat. no. C36950; Invitrogen; Thermo Fisher Scientific) were mixed with the BALF cells and assessed via flow cytometry. The numbers of BALF cells were calculated by comparing the ratio of beads events to cell events according to the manufacturer's protocol. The BALF cells were stained with antibodies against CD3-FITC (cat. no. 100203; Biolegend, Inc.), B220-FITC (cat. no. 103205, Biolegend, Inc.), CCR3-PE (cat. no. 144505; Biolegend, Inc.), CD11c-PerCP/Cyanine5.5 (cat. no. 117327; Biolegend, Inc.) and MHCII-APC (cat. no. 116417; BD Biosciences) for 30 min at 4°C. The cells were stained for neutrophils, mononuclear cells and lymphocytes (data not shown), and eosinophils using a CytoFLEX flow cytometer (Beckman Coulter, Inc.), and data collected were analyzed with FlowJo V10 software (FlowJo, LLC). Granulocytes were recognized as non-autofluorescent highly granular (SSChi) cells, and within this gate, eosinophils were defined as cells expressing the eotaxin receptor CCR3 and with low expression of MHCII, B220 and CD3. Neutrophils had a similar scatter profile as eosinophils but lacked CCR3 expression. Lymphocytes were identified as FSClo/SSClo and expressing CD3 or B220. Mononuclear cells expressed high levels of MHCII and CD11c.
10
Murine Immune Cell Characterization
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The reagents and antibodies used in this study were referenced from our previous reports28 (link)–30 (link),32 (link),60 (link). Lipopolysaccharide (LPS) from Escherichia coli (LPS O55:B5, L2880) was purchased from Sigma-Aldrich (MO, USA). Pam3csk4, CpG-ODNs and cGAMP (cyclic guanosine monophosphate–adenosine monophosphate) were purchased from Invivogen (CA, USA). Murine GM-CSF (granulocyte–macrophage colony-stimulating factor) was purchased from Peprotech (Rocky Hill, NJ, USA). The anti-mouse TLR7 mAb A94B10 and the anti-TLR9 mAb NaR9 were purified from ascitic fluid, as reported previously29 (link),30 (link). Streptavidin–phycoerythrin (PE), anti-mouse IgG1-PE, anti-mouse IgG2a-PE, isotype control antibodies (mouse IgG1, mouse IgG2a), and antibodies against mouse CD16/32, CD3-PE-Cy7, CD45-APC-Cy7, B220-FITC, CD11b-APC, CD11c-BV421, CD11c-PE-Cy7, Siglec-F-FITC, Ly-6G-FITC, CD4-BV510, CD8α-Percp/Cy5.5, CCR3-Percp/Cy5.5, I-A/I-E-BV510, CD103-FITC and IL17A-APC were purchased from BioLegend (San Diego, CA, USA). Anti-mouse IL5-PE, anti-mouse IL-2-APC, and anti-mouse IL13-eFluor450 were purchased from Invitrogen (Thermo Fisher Scientific, Minato-ku, Tokyo, Japan). Mouse IL-17A and IL-2 recombinant proteins were purchased from BioLegend.
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