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Anti b220

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Anti-B220 is a monoclonal antibody that binds to the B220 antigen, which is expressed on the surface of B lymphocytes. This antibody is commonly used in research applications for the identification and isolation of B cells.

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Lab products found in correlation

Anti cd4, by Thermo Fisher Scientific (15 mentions) Anti cd8, by Thermo Fisher Scientific (11 mentions) Anti cd11b, by Thermo Fisher Scientific (10 mentions) Anti ly6g, by Thermo Fisher Scientific (7 mentions) Anti cd11c, by Thermo Fisher Scientific (7 mentions) Anti cd3, by Thermo Fisher Scientific (7 mentions) Anti cd4, by BioLegend (6 mentions) Anti f4 80, by Thermo Fisher Scientific (6 mentions) Anti cd45, by Thermo Fisher Scientific (6 mentions) Anti gr 1, by Thermo Fisher Scientific (6 mentions) Triton x 100, by Merck Group (5 mentions) Anti nk1, by Thermo Fisher Scientific (5 mentions) Anti cd19, by Thermo Fisher Scientific (4 mentions) Anti cd45, by BioLegend (4 mentions) Anti cd16 32, by Thermo Fisher Scientific (4 mentions) Low melting agarose, by Thermo Fisher Scientific (4 mentions) Anti fcγ receptor, by BD (4 mentions) Anti ly6c, by Thermo Fisher Scientific (4 mentions) Anti foxp3, by Thermo Fisher Scientific (4 mentions) Anti cd44, by Thermo Fisher Scientific (4 mentions)

Spelling variants of this product

Anti-B220-APC (11 citations) Anti-mouse B220 (9 citations) Anti-B220 FITC (9 citations) PE-Cy7-conjugated anti-B220 (3 citations) Anti-B220-APC-eFluor 780 (2 citations) APC-Cy7–conjugated anti-B220, (2 citations) Anti-mouse B220-PE (2 citations) Anti-B220 APC-eFlour (2 citations) Anti-B220 eFluor 450 (2 citations) Anti-mouse B220-HorizonViolet500 (1 citations)

46 protocols using anti b220

1

Comprehensive Immune Cell Profiling

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BM cells, splenocytes, and peritoneal cells were stained with optimally diluted primary antibodies or isotype controls (30min 22°C), washed, and resuspended in PBS/0.5% BSA. Fifty thousand to 100,000 events per sample were acquired using a BD LSRII flow cytometer (BD Bioscience, San Jose, CA) and analyzed with FCS Express 3 (De Novo Software, Ontario, Canada). Anti-Ly6G, anti-B220, anti-CD11b, anti-B220, and anti-TNFα conjugated to allophycocyanin (APC), Pacific Blue (PB), fluorescein isothiocyanate (FITC), phycoerythrin (PE), or peridinin-chlorophyll-protein (PerCP) were obtained from eBioscience (San Diego, CA). Anti-CD138-APC and anti-CD19-PB were from BioLegend (San Diego, CA).
Zhuang H., Han S., Xu Y., Li Y., Wang H., Yang L.J, & Reeves W.H. (2014). Toll-like receptor 7-stimulated tumor necrosis factor α causes bone marrow damage in systemic lupus erythematosus. Arthritis & rheumatology (Hoboken, N.J.), 66(1), 140-151.
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2

Comprehensive Immune Cell Profiling in Mice

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Immune cells of 3-month-old male mice were isolated from spleen, kidney, bone marrow and thymus by flushing and pelleting (bone marrow) or by mechanical dissociation followed by centrifugation through a percoll gradient (kidney, spleen and thymus). Erythrocytes were lysed with ammonium-chloride-potassium lysing buffer, and the resulting leukocytes were counted on a haemocytometer. Cells were stained for flow cytometry analysis with anti-CD45 (Becton Dickinson, ref: 25-0451-82, dilution: 1/1,000), anti-B220 (eBioscience, ref: 45-0452-80, dilution: 1/1,000), anti-CD11b (eBioscience, ref: 12-0112-82, dilution: 1/1,000), anti-CD11c (eBioscience, ref: 11-0114-85, dilution: 1/1,000), anti-NKp46 (eBioscience, ref: 48-3351-82, dilution: 1/1,000), anti-CD8 (eBioscience, ref: 17-0081-81, dilution: 1/1,000), anti-TCRβ (Becton Dickinson, ref: 563221, dilution: 1/1,000) and Fixable Viability Dye (eBioscience, ref: 65-0865-14). Samples were acquired on CyAnADP 9 flow cytometer (Beckman Coulter) using Summit acquisition software (V). Data were analysed using Kaluza flow analysis software V (Beckman Coulter). An example of this analysis is shown in Supplementary Fig. 12.
Messaoudi S., He Y., Gutsol A., Wight A., Hébert R.L., Vilmundarson R.O., Makrigiannis A.P., Chalmers J., Hamet P., Tremblay J., McPherson R., Stewart A.F., Touyz R.M, & Nemer M. (2015). Endothelial Gata5 transcription factor regulates blood pressure. Nature Communications, 6, 8835.
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3

Multicolor Immunofluorescence Imaging of Lymph Nodes

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Skin draining LNs (axillary and brachial) were either freshly embedded in tissue freezing medium or fixed overnight in 2% paraformaldehyde/30% sucrose solution at 4°C and embedded in tissue freezing medium. 6–8 μm thick cryostat sections were cut for imaging by the fluorescence microscope. Primary antibodies used included biotinylated or purified anti-B220 (eBiosciences), anti-TCRβ (BD Biosciences), anti-LYVE-1 (Upstate), anti-CD31 (Serotec), anti-Ki67 (Dako), anti-CCL21 (R&D Systems), anti-collagen type IV (Cosmo Bio), FITC-conjugated anti-CD169 (Serotec), biotinylated anti-CD45.1 (eBiosciences) antibodies. Secondary antibodies used included Dylight647-conjugated streptavidin, Cy2 or Cy3-conjugated anti-rat IgG, Dylight647 or Dylight549-conjugated anti-armenian hamster IgG, Dylight 647-conjugated anti-goat IgG and Cy2, Cy3, or Dylight647-conjugated anti-rabbit IgG antibodies (Jackson Immunoresearch). Endogenous avidin and biotin were quenched using the Avidin/Biotin blocking kit (Vector Laboratories).
Tay M.H., Lim S.Y., Leong Y.F., Thiam C.H., Tan K.W., Torta F.T., Narayanaswamy P., Wenk M, & Angeli V. (2019). Halted Lymphocyte Egress via Efferent Lymph Contributes to Lymph Node Hypertrophy During Hypercholesterolemia. Frontiers in Immunology, 10, 575.
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4

Multicolor Flow Cytometry Panel for Immune Cell Profiling

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Cells were stained with the following fluorochrome-conjugated antibodies: anti-B220 (eBioscience, clone RA3-6B2), anti-CD3e (eBioscience, clone 145-2C11), anti-CD8a (BD, clone 53-6.7), anti-CD11b (eBioscience, clone M1/70), anti-CD11c (Bio Legend, clone N418), anti-CD14 (Bio Legend, clone SA14-2), anti-CD19 (eBioscience, clone eBio1D3), anti-CD64 (Bio Legend, clone X54-5/7.1), anti-CD68 (AbD Serotec, clone FA-11), anti-CD163 (Bioss, polyclonal), anti-CD115 (eBioscience, clone AF598), anti-CCR3 (Bio Legend, clone J073E5), anti-F4/80 (Bio Legend, clone CI:A3-1), anti-FPR-1 (Bioss, polyclonal), anti-MHC II (Bio Legend, clone M5/114.15.2), anti-MR (AbD Serotec, clone MR5D3) and anti-PILRa (R&D Systems, polyclonal). Fc receptors were blocked with 1.5mg/ml human IgG (Privigen). Dead cells were excluded using the Hoechst 33342 dye. Only events that appeared single in forward-scatter width were analyzed. The gating strategy is shown in Supplementary Figure 7. A FACSCanto II and FACSDiva software (BD) were used for flow cytometry and data were analyzed with FlowJo software (TreeStar).
Franken L., Klein M., Spasova M., Elsukova A., Wiedwald U., Welz M., Knolle P., Farle M., Limmer A, & Kurts C. (2015). Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates. Scientific Reports, 5, 12940.
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5

Isolation and Flow Cytometry of Neutrophils

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Flow cytometry analysis was performed on a LSRII or LSRFortessa (BD Biosciences). To isolate neutrophils from the feet, tissue was diced and incubated at 37°C for 45 min with shaking at 200 rpm (TissueLyserII, Qiagen), in DMEM containing 1 mg/mL Collagenase Type IV (Worthington Biochemical). Cells were then strained through a 70 μM strainer and washed twice in phosphate buffered saline (PBS). Neutrophils from bone marrow and feet were sorted on a FACSAria III (BD Biosciences). FcγR were blocked using TruStain FcX (BioLegend). Data were analyzed using FlowJo software (BD Biosciences). The following reagents were used: Zombie-NIR fixable viability kit (BioLegend), anti-B220 (eBioscience;RA36B2), anti-CD3 (WEHI;KT3.1.1), anti-CD4 (WEHI;GK1.5), anti-CD8 (WEHI;53.6.7), anti-CD11b (BioLegend;M1/70), anti-CD16/32 (WEHI;24G2), anti-CD34 (eBioscience;RAM34), anti-CD45.1 (BioLegend;A20), anti-CD45.2 (BioLegend;104), anti-CD48 (BD Bioscience;C2), anti-CD127 (eBioscience;A7R34), anti-CD135 (BioLegend;A2F10), anti-CD150 (BioLegend;TC15–12F12.2), anti-cKit (WEHI;ACK4), anti-Gr1 (WEHI;RB6–8C5), anti-Ly6G (BD;1A8), anti-Sca-1 (WEHI;Ly6A/E), anti-Siglec-F (Fisher;1RMN44N), AnnexinV-AF647 (BioLegend), and propidium iodide (Sigma).
Speir M., Nowell C.J., Chen A.A., O’Donnell J.A., Shamie I.S., Lakin P.R., D’Cruz A.A., Braun R.O., Babon J.J., Lewis R.S., Bliss-Moreau M., Shlomovitz I., Wang S., Cengia L.H., Stoica A.I., Hakem R., Kelliher M.A., O’Reilly L.A., Patsiouras H., Lawlor K.E., Weller E., Lewis N.E., Roberts A.W., Gerlic M, & Croker B.A. (2019). Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation. Nature immunology, 21(1), 54-64.
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6

Immunohistochemistry of Lymph Nodes

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Immunohistochemistry was performed using a described method (31 (link)). Freshly isolated LNs were fixed in freshly prepared 4% paraformaldehyde (Electron Microscopy Science) and agitated overnight at 4°C. LNs were embedded in 4% low melting agarose (Invitrogen) in PBS and sectioned with a vibratome (Leica VT-1000 S) at a 30 µm thickness. Thick sections were blocked in PBS containing 10% FCS, 1 mg/ml anti-Fcγ receptor (BD Biosciences), and 0.1% Triton X-100 (Sigma) for 30 minutes at room temperature. Sections were immunostained with the following antibodies: anti-B220, anti-CD4, anti-Ki67 (all from eBioscience), anti-CD169 (R&D System), anti-CD21/35 (BioLegend) and agitated overnight at 4°C. Stained thick sections were microscopically analyzed by using a Leica SP5 confocal microscope (Leica Microsystem, Inc.) and images were processed with Leica LAS AF software (Leica Microsystem, Inc.) and Imaris v.7.6.1 (Bitplane).
Hwang I.Y., Park C., Harrison K., Boularan C., Galés C, & Kehrl J.H. (2015). An Essential Role for RGS Protein/Gαi2 Interactions in B Lymphocyte Directed Cell Migration and Trafficking. Journal of immunology (Baltimore, Md. : 1950), 194(5), 2128-2139.
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7

Isolation and Flow Cytometry of Neutrophils

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Flow cytometry analysis was performed on a LSRII or LSRFortessa (BD Biosciences). To isolate neutrophils from the feet, tissue was diced and incubated at 37°C for 45 min with shaking at 200 rpm (TissueLyserII, Qiagen), in DMEM containing 1 mg/mL Collagenase Type IV (Worthington Biochemical). Cells were then strained through a 70 μM strainer and washed twice in phosphate buffered saline (PBS). Neutrophils from bone marrow and feet were sorted on a FACSAria III (BD Biosciences). FcγR were blocked using TruStain FcX (BioLegend). Data were analyzed using FlowJo software (BD Biosciences). The following reagents were used: Zombie-NIR fixable viability kit (BioLegend), anti-B220 (eBioscience;RA36B2), anti-CD3 (WEHI;KT3.1.1), anti-CD4 (WEHI;GK1.5), anti-CD8 (WEHI;53.6.7), anti-CD11b (BioLegend;M1/70), anti-CD16/32 (WEHI;24G2), anti-CD34 (eBioscience;RAM34), anti-CD45.1 (BioLegend;A20), anti-CD45.2 (BioLegend;104), anti-CD48 (BD Bioscience;C2), anti-CD127 (eBioscience;A7R34), anti-CD135 (BioLegend;A2F10), anti-CD150 (BioLegend;TC15–12F12.2), anti-cKit (WEHI;ACK4), anti-Gr1 (WEHI;RB6–8C5), anti-Ly6G (BD;1A8), anti-Sca-1 (WEHI;Ly6A/E), anti-Siglec-F (Fisher;1RMN44N), AnnexinV-AF647 (BioLegend), and propidium iodide (Sigma).
Speir M., Nowell C.J., Chen A.A., O’Donnell J.A., Shamie I.S., Lakin P.R., D’Cruz A.A., Braun R.O., Babon J.J., Lewis R.S., Bliss-Moreau M., Shlomovitz I., Wang S., Cengia L.H., Stoica A.I., Hakem R., Kelliher M.A., O’Reilly L.A., Patsiouras H., Lawlor K.E., Weller E., Lewis N.E., Roberts A.W., Gerlic M, & Croker B.A. (2019). Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation. Nature immunology, 21(1), 54-64.
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8

Splenocyte Isolation and Flow Cytometry

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Single-cell suspensions of splenocytes were prepared, depleted of red blood cells by hypotonic lysis, and stained with optimal dilutions of the indicated antibodies. All of the following reagents were obtained from eBioscience: anti-CD4 (RM4–5), anti-CD8a (53–6.7), anti-Gr-1 (RB6–8C5), anti-F4/80 (BM8), and anti-TER119; anti-IgD (11–26); anti-B220 (RA3–6B2); anti-CD19 (1D3). Doublets were excluded using the combined width and height parameters of the forward and side scatter parameters. Flow cytometric acquisition was performed on a BD LSRII, and analyses were performed using FlowJo 10.1r5 (Tree Star). Cells were sorted with a three-laser FACsAria Fusion.
Bortnick A., He Z., Aubrey M., Chandra V., Denholtz M., Chen K., Lin Y.C, & Murre C. (2020). Plasma Cell Fate Is Orchestrated by Elaborate Changes in Genome Compartmentalization and Inter-chromosomal Hubs. Cell reports, 31(1), 107470.
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9

Comprehensive Immune Cell Analysis

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Anti–mouse CD4, anti–mouse T-cell receptor (TCR) β, Anti–mouse CD44, anti–mouse CD62L, anti-mouse CD25, anti–mouse Foxp3, anti-CD8, and anti–B220 antibodies were purchased from eBio-science (San Diego, CA). Anti-mouse CD138 antibody was purchased from BD Biosciences (Franklin Lakes, NJ).
Chen J., Schroeder J.A., Luo X, & Shi Q. (2017). The impact of von Willebrand factor on factor VIII memory immune responses. Blood Advances, 1(19), 1565-1574.
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10

Isolation and Analysis of Germinal Center B Cells

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Spleens were collected from WT mice and cells were resuspended in staining buffer (5% FBS in PBS). Red blood cells (RBC) were lysed using ACK lysis buffer (Lonza). To isolate GCBCs, single-cell suspensions from the spleens of immunized mice were labeled with biotinylated antibodies specific for CD43, CD11c and IgD followed by depletion using standard magnetic bead technology (Miltenyi magnetic columns). The resulting unlabeled cells were enriched for GCBCs. Enriched cells were then stained with anti-B220, anti-GL7, anti-CD95, anti-CXCR4, anti-CD83, and viability dye eFluor 506 (eBioscience). GCBC subsets were then isolated by fluorescence activated cell sorting (FACS). For cell cycle analysis cells were stained with FxCycle Violet (ThermoFisher). Cell sorting was performed on a FACSAria II (BD) and flow cytometry was performed on an LSR Fortessa X20 (BD). Flow cryometric analysis was performed using FlowJo (BD).
Kennedy D.E., Okoreeh M.K., Maienschein-Cline M., Ai J., Veselits M., McLean K.C., Dhugana Y., Wang H., Peng J., Chi H., Mandal M, & Clark M.R. (2020). Novel specialized cell state and spatial compartments within the germinal center. Nature immunology, 21(6), 660-670.
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