B220 apc

Manufactured by BioLegend

Sourced in United States

B220-APC is a fluorochrome-conjugated monoclonal antibody targeting the B220 antigen, also known as CD45R. B220 is a high molecular weight isoform of the CD45 family of tyrosine phosphatases, which is expressed on the surface of B cells. This antibody can be used for the identification and enumeration of B cells in flow cytometry applications.

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23 protocols using b220 apc

Comprehensive Analysis of Immune Cell Subsets in Rag1 Mice

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Total cellularity of thymus, spleen, bone marrow and the distribution of the various T and B cell subsets were analyzed in 8-12 week-old ΔRag1 mice and Rag1 H836Q mice. Both groups were compared to age-matched wild type (wt) and Rag1 ^−/− mice (Jackson Laboratory) [19 (link)]. Splenocytes were stained with a T cell panel consisting of CD4-PB (Ebioscience), CD8-PE/Cy5 (BD), CD3 PE-Cy7 (Biolegend) and a B cell panel consisting of B220-APC (Biolegend), IgM-PE-cy5 (Ebioscience), CD43-PE (Ebioscience). Bone marrow cells were stained with B220-APC (Biolegend), IgM-PE-cy5 (Ebioscience), and CD43-PE (Ebioscience) antibodies. Thymocytes were stained with CD3-PE-Cy7 (Biolegend), CD4-PB (Ebioscience), CD8-PE-Cy5 (BD), CD44-FITC (BD), and CD25-PE (Ebioscience) antibodies, upon excluding B220 (Biolegend), Ter119 (Biolegend) and MAC1 (Biolegend) positive cells. FACS gating strategies are shown in Supplementary Figures S1 to S4 (S1-S4). Standard FSC/SSC live gates and FSC/SSC lymphocyte gates were used.

de Bruin L.O., Yang W., Capuder K., Lee Y.N., Antolini M., Meyers R., Gellert M., Musunuru K., Manis J, & Notarangelo L. (2016). Rapid generation of novel models of RAG1 deficiency by CRISPR/Cas9-induced mutagenesis in murine zygotes. Oncotarget, 7(11), 12962-12974.

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Sequencing of Murine Somatic Hypermutation

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Mice were immunised orally with sheep red blood cells for 2 weeks and intraperitoneally with 10 μg of LPS for 3 days. Single-cell suspensions from Peyer's patches were labelled with B220-APC- (Biolegend, ref:103212), GL7-FITC- (Beckton Dikinson, ref: 561530) and Fas-PE- (Beckton Dikinson, ref: 554258) conjugated antibodies. Purification of B220⁺GL7⁺Fas⁺ cells was realized on a FACS ARIA III (BD). Genomic DNA was extracted, and a region corresponding to a sequence of 517 bp downstream of the J_H4 segment was amplified by PCR. As a control, Igκ light-chain VJ-rearranged fragments were also amplified. Primers (detailed in the Supplementary Table 1) were coupled to 454 Sequencing adaptor sequences and PCR was performed using the program previously reported8 (link). According to the manufacturer, the resulting purified amplicons were prepared for sequencing with a GS Junior Titanium emPCR Kit (Lib-A; Roche), and the library of DNA fragments was sequenced on a 454 GS Junior instrument (Roche). Obtained sequences were aligned to the reference sequence using BWA aligner38 , and SAMtools software was used to obtain BAM files39 (link). Redundant sequences were excluded, and wig files were generated using IGV Tools40 (link) and manually analysed to determine mutation frequencies for each nucleotide in the sequence.

Saintamand A., Vincent-Fabert C., Garot A., Rouaud P., Oruc Z., Magnone V., Cogné M, & Denizot Y. (2016). Deciphering the importance of the palindromic architecture of the immunoglobulin heavy-chain 3' regulatory region. Nature Communications, 7, 10730.

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Multiparametric Flow Cytometry Analysis

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Single cell suspension of splenocytes, lymph node, and peritoneal lavage were incubated with 10 μg anti-mouse unlabeled CD16/32 (2.4G2); stained with B220-APC or PeCy5, CD3-PE or PECy7, CD11b-FITC, or GL7-FITC (Biolegend), washed, examined on a BD Canto Flow analyzer, and data analyzed with FCS Express, v. 4.

Cooley L.F., El Shikh M.E., Li W., Keim R.C., Zhang Z., Strauss J.F., Zhang Z, & Conrad D.H. (2016). Impaired immunological synapse in sperm associated antigen 6 (SPAG6) deficient mice. Scientific Reports, 6, 25840.

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Murine Immune Cell Characterization

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Sex matched mice at the age of 6–8 weeks were sacrificed by CO₂ asphyxiation. Peritoneal cavity cells were collected by rinsing with FACS buffer (1x PBS plus 2% fetal bovine serum). Cells from the bone marrow and spleen were lysed with a red blood cell lysis buffer to remove red blood cells and filtered through a 70 μM cell strainer (BD Biosciences, Franklin Lakes, NJ). After washing with cold 1x PBS, cells were counted by trypan blue staining. One million cells were resuspended in 100 μL FACS buffer and labeled with 1 μL antibodies of B220-APC, CD43-FITC, BP-1-PE, CD24-PerCP/Cy5.5, CD19-APC-Cy7, IgM-PE, IgD-PerCP/Cy5.5, IgM-PErCP/Cy5.5, CD93-APC, CD23-PE, CD21-FITC, CD5-PerCP (BioLegend, San Diego, CA). Cell subpopulations were analyzed on BD LSR II violet at The University of Iowa flow cytometry facility.

Gu Z., Zhou W., Huang J., Yang Y., Wendlandt E., Xu H., He X., Tricot G, & Zhan F. (2014). Nek2 Is a Novel Regulator of B Cell Development and Immunological Response. BioMed Research International, 2014, 621082.

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Multicolor Immunofluorescence Staining of Mouse Spleen

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Fresh spleens were embedded in optimal cutting temperature (OCT; TissueTek) and frozen on dry ice. After cryostat sectioning at 6-μm, spleens were fixed with 4% PFA for 20 minutes and treated with 3% Hydrogen Peroxide for 30 minutes before blocking with an Avidin/Biotin Vector Labs Blocking Kit and 2% Fetal Bovine Serum. Primary antibody incubation included CD4-FITC (BD Biosciences), B220-APC (Biolegend), and Thy1.1-Biotin (Biolegend; clone OX-7). After primary antibody incubation, sections were washed in PBS before secondary amplification was performed using streptavidin (SA)-Rhodamine Red-X (Jackson Immuno Research Labs). For additional rounds of secondary amplification, sections were washed with PBS before incubation with biotinylated anti-SA (Vector Labs), washed, and stained again with SA-Rhodamine Red-X. The amplification process was repeated once more for a total of 3 amplifications with Rhodamine Red-X. Sections were visualized using the Zeiss Axio Observe Z1 immunofluorescence microscope (Carl Zeiss, Inc.) and images were taken using an AxioCam MRm camera with a 10x objective (Carl Zeiss, Inc.). Images were prepared using Adobe Photoshop.

Cunningham C.R., Champhekar A., Tullius M.V., Dillon B.J., Zhen A., de la Fuente J.R., Herskovitz J., Elsaesser H., Snell L.M., Wilson E.B., de la Torre J.C., Kitchen S.G., Horwitz M.A., Bensinger S.J., Smale S.T, & Brooks D.G. (2016). Type I and Type II Interferon Coordinately Regulate Suppressive Dendritic Cell Fate and Function during Viral Persistence. PLoS Pathogens, 12(1), e1005356.

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Multi-parameter Immune Cell Profiling

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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).

Tejon G., Valdivieso N., Flores-Santibañez F., Barra-Valdebenito V., Martínez V., Rosemblatt M., Sauma D, & Bono M.R. (2022). Phenotypic and functional alterations of peritoneal macrophages in lupus-prone mice. Molecular Biology Reports, 49(6), 4193-4204.

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Surface Marker Profiling of Immune Cells

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Surface staining of BMC and spleen cells previously treated with erythrocyte lysis buffer (Qiagen) was performed with antibodies from BD Pharmingen against CD3-PE-Cy7, CD4-APC, CD8-PB, B220-APC, Ter119-PE-Cy7, CD11b-PE-Cy7, CD19-PE-Cy7, Gr-1-PE-Cy7, Gr-1-PB and Sca1-PB; cKit-APC from BioLegend (San Diego, CA, USA), and CD90.2-PE-Cy7 from eBioscience (San Diego, CA, USA) and measured using the CyanADP flow cytometer (Beckmann Coulter). Flow cytometry data were analyzed using the FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA).

Sprissler C., Belenki D., Maurer H., Aumann K., Pfeifer D., Klein C., Müller T.A., Kissel S., Hülsdünker J., Alexandrovski J., Brummer T., Jumaa H., Duyster J, & Dierks C. (2014). Depletion of STAT5 blocks TEL–SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice. Blood Cancer Journal, 4(8), e240-.

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Phenotyping Fungal Lung Infection

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C57BL/6 mice (Jackson) were infected with 10⁶ Uvitex 2B-labeled (10 µg/ml; 5 min) yeast cells administered intratracheally. Single cell suspensions were made from harvested lungs with a 70-µm cell strainer and treatment with collagenase D (1 mg/ml; Roche) and DNase (50 U/ml; Roche). Leukocytes were enriched by density sedimentation (60%/40% Percoll, 20 min, relative centrifugal force of 600) and collection of cells at the interface. Approximately 10⁶ cells were stained for cellular markers and fixed with 4% paraformaldehyde (30 min). The markers included CD64-fluorescein isothiocyanate, CD45-peridinin chlorophyll protein-Cy5.5, SiglecF-phycoerythrin-Cy7, Ly6C-BV650, CD11c-BV786, CD90.2-allophycocyanin (APC), B220-APC, MHCII-A700, Ly6G-BUV395 (all from BioLegend), and Near IR live/dead stain (Thermo, Fisher). Cells were analyzed with an LSRII flow cytometer (BD Biosciences), and data were processed with FlowJo software (version 10.1).

Garfoot A.L., Goughenour K.D., Wüthrich M., Rajaram M.V., Schlesinger L.S., Klein B.S, & Rappleye C.A. (2018). O-Mannosylation of Proteins Enables Histoplasma Yeast Survival at Mammalian Body Temperatures. mBio, 9(1), e02121-17.

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Multicolor Flow Cytometry Panel

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The following antibodies were used: mouse lineage cocktail-PE (BioLegend, cat# 78035), mouse lineage cocktail-APC (R&D Systems, cat# FLC001A), c-Kit-FITC (BioLegend, cat# 161603), c-Kit-APC (BioLegend, cat# 135108), c-Kit-PE/Cy7 (BioLegend, cat# 105814), c-Kit-BV421 (BioLegend, cat# 135124), Sca-1-BV605 (BioLegend, cat# 108133), Sca-1-APC (eBioscience, cat# 17-5981-82), Sca-1-BV421 (BioLegend, cat# 108127), Sca-1-PerCP-Cy5.5 (eBioscience, cat# 45-5981-82), CD150-BV421 (BioLegend, cat# 115925), CD150-PE (eBioscience, cat# 12-1501-82), CD150-PE-Cy7 (BioLegend, cat# 115913), CD48-PE/Cy7 (eBioscience, cat# 25-0481-80), CD48-BV711 (BioLegend, cat# 103439), CD48-APC-Cy7 (BioLegend, cat# 103432),CD34-FITC (eBioscience, cat# 11-0341-82), CD135-PE-Cy5 (BioLegend, cat# 135311), CD135-APC (BioLegend, cat# 135310), CD45.1-PerCP-Cy5.5 (BioLegend, cat# 110727), CD45.2-BV421 (BioLegend, cat# 109831), CD45.2-FITC(eBioscience, cat# 11-0454-82), Gr1-PE (BioLegend, cat# 108407), CD11b-APC (BioLegend cat# 101211), CD4-PE (BioLegend,cat# 116005), CD8a-PE (BioLegend, cat# 100707), B220-APC (BioLegend, cat# 103211).

Wang S., Han J., Huang J., Islam K., Shi Y., Zhou Y., Kim D., Zhou J., Lian Z., Liu Y, & Huang J. (2024). Deep learning-based predictive classification of functional subpopulations of hematopoietic stem cells and multipotent progenitors. Stem Cell Research & Therapy, 15, 74.

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Multiparametric Immune Cell Profiling

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MHC class II tetramer-PE (from the NIH tetramer core facility at Emory, Atlanta, GA), CD4 BUV737 (BD, clone GK1.1), CD8 PerCP-Cy5.5 (Biolegend, clone 53–6.7, cat 100734), CD44 BV785 (Biolegend, clone IM7), CD11b APC (Biolegend, clone M1/70, cat 101212), CD11c APC (Biolegend, clone N418, cat 117310), NK1.1 APC (Biolegend, clone PK136, cat 108710), B220 APC (Biolegend, clone RA3-62B, cat 103212).

dos Santos Dias L., Dobson H.E., Bakke B.K., Kujoth G.C., Huang J., Kohn E.M., Taira C.L., Wang H., Supekar N.T., Fites J.S., Gates D., Gomez C.L., Specht C.A., Levitz S.M., Azadi P., Li L., Suresh M., Klein B.S, & Wüthrich M. (2021). Structural basis of Blastomyces Endoglucanase-2 adjuvancy in anti-fungal and -viral immunity. PLoS Pathogens, 17(3), e1009324.

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