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B220 apc cy7

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B220-APC/Cy7 is a fluorochrome-conjugated antibody that binds to the B220 antigen, also known as CD45R, which is expressed on the surface of B cells. It is a useful tool for the identification and analysis of B cell populations in flow cytometry applications.

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Lab products found in correlation

Lsrii flow cytometer, by BD (3 mentions) Ly6c fitc, by BioLegend (3 mentions) Cd45 percp cy5, by BioLegend (3 mentions) Nk1.1 apc cy7, by BioLegend (3 mentions) Facsaria 2, by BD (2 mentions) Ly6g pe cy7, by BioLegend (2 mentions) Cd3 apc cy7, by BioLegend (2 mentions) Cd19 apc cy7, by BioLegend (2 mentions) Cd11b bv510, by BioLegend (2 mentions) Cd11b apc, by BioLegend (2 mentions) Hyaluronidase, by Merck Group (2 mentions) Dnase, by Merck Group (2 mentions) Collagenase 1, by Merck Group (2 mentions) Collagenase xi, by Merck Group (2 mentions) Rneasy plus micro kit, by Qiagen (2 mentions) Cd64 apc, by BioLegend (2 mentions) Cd115 bv605, by BioLegend (2 mentions) Cd45 bv711, by BioLegend (2 mentions) Ly6g pe, by BioLegend (2 mentions) Mac1 pecy7, by BioLegend (2 mentions)

Spelling variants of this product

APC-Cy7 (106 citations) B220-APC (23 citations) Anti-B220 APC-cy7 (10 citations) B220-APC and CD11b-APC/cy7 antibodies (3 citations) B220-APC-Cy7 (RA3-6B2; (2 citations)

13 protocols using b220 apc cy7

1

Multicolor Flow Cytometry for Immune Cell Profiling

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PE anti-CD3, Pacific Blue anti-CD8α, biotin-conjugated anti-CD8β, PE Cy7 anti-CD4, APC Cy7 anti-CD62L, APC anti-CD103, PerCP Cy5.5 anti-TCRγδ, PE Cy7 anti-TCRβ, APC Cy7-B220, PerCP Cy5.5 anti-B220, FITC-EpCAM, PE Cy7 anti-CD11b, APC anti-F4/80, biotin-conjugated anti-IgA antibodies were purchased from BioLegend. Streptavidin-PE was purchased from BD Biosciences.
Ito K., Nakajima A., Fukushima Y., Suzuki K., Sakamoto K., Hamazaki Y., Ogasawara K., Minato N, & Hattori M. (2017). The potential role of Osteopontin in the maintenance of commensal bacteria homeostasis in the intestine. PLoS ONE, 12(3), e0173629.
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2

Isolation and Immunophenotyping of Mouse Hematopoietic Cells

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Bone marrow and spleen of a transplant recipient mouse were harvested, and single cell suspensions were prepared as previously described34 (link). Briefly, the femurs and tibias were dissected using sterile technique, flushed with PBS/5% FBS, and cells were passed through a 70 μm strainer. Spleens were dissected cleanly using sterile technique, placed on a 70 μm strainer and using the plunger end of a syringe, gently passed through the strainer. Red blood cells were lysed using RBC lysis buffer (Biolegends, San Diego, California). Cells were resuspended in PBS/5% FBS, and then counted using a Cellometer Mini cell counter (Nexcelom, Lawrence, MA). One million cells of each sample were then distributed into microcentrifuge tubes and labelled with fluorochrome-conjugated antibodies APC/Cy7-B220, FITC-Ter119, PE/Cy7-Gr1, and PerCP/Cy5.5-CD11b (all antibodies were purchased from Biolegends and added at predetermined optimum concentrations). Immunophenotyping was performed in the UVA Flow Cytometry core lab using a Fortessa cytometer, and data were analyzed using the FlowJo program (FlowJo LLC, Ashland, Oregon).
Hu Y., Belyea B.C., Li M., Göthert J.R., Gomez R.A, & Sequeira-Lopez M.L. (2017). Identification of cardiac hemo-vascular precursors and their requirement of sphingosine-1-phosphate receptor 1 for heart development. Scientific Reports, 7, 45205.
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3

Characterizing Cardiac Immune Populations

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Heart tissue was minced and enzymatically digested using collagenase I (450 U ml−1), collagenase XI (60 U ml−1), DNAse and hyaluronidase (60 U ml−1) (Sigma-Aldrich). Single-cell suspensions were stained with CD45-PerCP/Cy5.5 (clone 30-F11, 1:600, 103132, BioLegend), CD64-APC (clone X54–5/7.1, 1:600, 139305, BioLegend), Ly6G-PE/Cy7 (clone 1A8, 1:600, 127617, BioLegend), Ly6C-FITC (clone HK1.4, 1:600, 128006, BioLegend), CD11b-BV510 (clone M1/70, 1:600, 101245, BioLegend) and DAPI (0.1%, F10347, Thermo Fisher Scientific). Blood samples from Ly6GTdtomato mice were stained with Ly6G-PE/Cy7 (clone 1A8, 1:600, 127617, BioLegend), CD115-BV605 (clone AFS98, 1:600, 135517, BioLegend), CD11b-APC (clone M1/70, 1:600, 101212, BioLegend), CD45-BV711 (30-F11, 1:600, 103147, BioLegend), CD3-APC/Cy7 (clone 17A2, 1:200, 100221, BioLegend), CD19-APC/Cy7 (clone 6D5, 1:300, 115529, BioLegend), B220-APC/Cy7 (clone RA3–6B2, 1:300, 103224, BioLegend), Nk1.1-APC/Cy7 (clone PK136, 1:300, 108724, BioLegend) and DAPI. Data were recorded on an LSRII flow cytometer with FACSDiva 6.1 and analyzed with FlowJo 10 software (BD Biosciences). For qRT–PCR measurements, cells were flow sorted on a FACSAria II (BD Biosciences) into 350 μl of lysis buffer (RNeasy Plus Micro Kit, Qiagen).
Grune J., Lewis A.J., Yamazoe M., Hulsmans M., Rohde D., Xiao L., Zhang S., Ott C., Calcagno D.M., Zhou Y., Timm K., Shanmuganathan M., Pulous F.E., Schloss M.J., Foy B.H., Capen D., Vinegoni C., Wojtkiewicz G.R., Iwamoto Y., Grune T., Brown D., Higgins J., Ferreira V.M., Herring N., Channon K.M., Neubauer S., Sosnovik D.E., Milan D.J., Swirski F.K., King K.R., Aguirre A.D., Ellinor P.T, & Nahrendorf M. (2022). Neutrophils incite and macrophages avert electrical storm after myocardial infarction. Nature cardiovascular research, 1(7), 649-664.
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4

Engraftment and Myeloid Cell Differentiation

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To evaluate engraftment and myeloid cell differentiation, peripheral blood cells were prepared and analyzed as previously described. Fluorescence-labeled CD45.2-APC (Cat. #109814), CD45.1-PE (Cat. #110708), Gr1-FITC (Cat. #108406), B220-APC/Cy7 (Cat. #103224), and CD3-PE/Cy7 (Cat. #1000320) were purchased from Biolegend, Inc. (San Diego, CA, USA), and DAPI was purchased from BD Biosciences (San Jose, CA, USA). These fluorescent antibodies were used to sort subsets of myeloid cells with a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) [21 (link),22 (link)].
Bei T., Cao X., Liu Y., Li J., Luo H., Huang L., Tian T., Li L, & Jiang Y. (2021). Antioxidant Fusion Protein SOD1-Tat Increases the Engraftment Efficiency of Total Bone Marrow Cells in Irradiated Mice. Molecules, 26(11), 3395.
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5

Multicolor Flow Cytometry Panel

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Commercial antibodies (clones, fluorophores and sources) are as follows: CD3-FITC (145–2C11), CD62L-PE (Mel-14), CD4-PErCP/Cy5.5 (RM4–5), CD8-PerCP/Cy5.5 (53–6.7), CD8-APC (53–6.7), CD209b-APC (22D1), IgM-FITC (II/41), CD4-PE (GK1.5), CD25-AF488 (PC61.5), Streptavidin-PE, CD169-PE/Cy7 (3D6.112) (eBioscience, San Diego, CA); CD11b-FITC (M1/70), CD49d-FITC (R1–2), TCRβ-FITC (GL3), CD21/35-PE (7E9), CD23-APC (B3B4), Ly6G-PE (RB6–8C5), Ly6C-PerCP/Cy5.5 (HK1.4), CD11c-PE/Cy7 (N418), B220-PerCP/Cy5.5 (RA3–6B2), B220-APC (RA3–6B2), B220-APC/Cy7 (RA3–6B2), CD69-BV421 (H1.2F3),CD23-Biotin (B3B4), F4/80-APC (BM8), Ly6G-BV421 (1A8), CD45-Pacific Blue (30-F11), CD45-BV510 (30-F11), CD43-PE (1B11), CD24-PE/Cy7 (M1/69), IgD-Pacific Blue (11–26c.2a), CD69-PE/Cy7 (H1.2F3), IgD-PerCP/Cy5.5 (11–26c.2a), Ly51-AF647 (6C3), CD3-Pacific Blue (17A2), CD3-PE (17A2), TCRγ/δ-biotin (GL3), Streptavidin-PE/Cy7 (Biolegend, San Diego, CA); Siglec-F-PE (E50–2440) (BD Biosciences, San Jose, CA). Samples were preincubated with 1 μg Fc-block (2.4G2 hybridoma; ATCC).
Cells were acquired either on the BD Biosciences LSR Fortessa or with a BD FACScan flow cytometer with DxP multi-color upgrades by Cytek Development Inc. (Woodland Park, NJ) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR).
Anaya E.P., Lin X., Todd E.M., Szasz T.P, & Morley S.C. (2021). Novel mouse model reveals that serine phosphorylation of L-plastin is essential for effective splenic clearance of pneumococcus. Journal of immunology (Baltimore, Md. : 1950), 206(9), 2135-2145.
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6

Characterizing Cardiac Immune Populations

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Heart tissue was minced and enzymatically digested using collagenase I (450 U ml−1), collagenase XI (60 U ml−1), DNAse and hyaluronidase (60 U ml−1) (Sigma-Aldrich). Single-cell suspensions were stained with CD45-PerCP/Cy5.5 (clone 30-F11, 1:600, 103132, BioLegend), CD64-APC (clone X54–5/7.1, 1:600, 139305, BioLegend), Ly6G-PE/Cy7 (clone 1A8, 1:600, 127617, BioLegend), Ly6C-FITC (clone HK1.4, 1:600, 128006, BioLegend), CD11b-BV510 (clone M1/70, 1:600, 101245, BioLegend) and DAPI (0.1%, F10347, Thermo Fisher Scientific). Blood samples from Ly6GTdtomato mice were stained with Ly6G-PE/Cy7 (clone 1A8, 1:600, 127617, BioLegend), CD115-BV605 (clone AFS98, 1:600, 135517, BioLegend), CD11b-APC (clone M1/70, 1:600, 101212, BioLegend), CD45-BV711 (30-F11, 1:600, 103147, BioLegend), CD3-APC/Cy7 (clone 17A2, 1:200, 100221, BioLegend), CD19-APC/Cy7 (clone 6D5, 1:300, 115529, BioLegend), B220-APC/Cy7 (clone RA3–6B2, 1:300, 103224, BioLegend), Nk1.1-APC/Cy7 (clone PK136, 1:300, 108724, BioLegend) and DAPI. Data were recorded on an LSRII flow cytometer with FACSDiva 6.1 and analyzed with FlowJo 10 software (BD Biosciences). For qRT–PCR measurements, cells were flow sorted on a FACSAria II (BD Biosciences) into 350 μl of lysis buffer (RNeasy Plus Micro Kit, Qiagen).
Grune J., Lewis A.J., Yamazoe M., Hulsmans M., Rohde D., Xiao L., Zhang S., Ott C., Calcagno D.M., Zhou Y., Timm K., Shanmuganathan M., Pulous F.E., Schloss M.J., Foy B.H., Capen D., Vinegoni C., Wojtkiewicz G.R., Iwamoto Y., Grune T., Brown D., Higgins J., Ferreira V.M., Herring N., Channon K.M., Neubauer S., Sosnovik D.E., Milan D.J., Swirski F.K., King K.R., Aguirre A.D., Ellinor P.T, & Nahrendorf M. (2022). Neutrophils incite and macrophages avert electrical storm after myocardial infarction. Nature cardiovascular research, 1(7), 649-664.
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7

Comprehensive Immune Cell Profiling

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We used the following gating schemes: total leukocytes (CD45.2+), total B cells (CD19+ CD3−), B-2 (CD19+ CD3− B220hi CD5− CD23+), B-1a (CD19+ CD3− IgM+ IgD− B220lo CD5+ CD23−), B-1b (CD19+ CD3− IgM+ IgD− B220lo CD5− CD23−), regulatory B [Breg] cells (CD19+ B220+ CD22+ CD5− IgM+ IgD+), total T cells (CD19− CD3+), Treg cells (CD19− CD3+ CD4+ CD25+), and macrophages (CD19− CD3− CD11b+ F4/80+). Dead cells were distinguished by Live/Dead Fixable Aqua staining (Life Technologies). Baselines for IL-10 EGFP mice were set using age- and diet-matched C57BL/6J mice. For macrophage intracellular cytokine staining, cells were stimulated with LPS (1 μg/mL; Sigma-Aldrich) and brefeldin A (5 μg/mL; BioLegend) overnight and stained using the Cytofix/Cytoperm Kit (BD Biosciences) according to the vendors’ instructions. Data were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).
CD16/32, CD3-PacBlue, CD5-PE-Cy5, CD19-PerCP-Cy5.5, CD22-PE, CD23-PE-Cy7, CD25-PE, CD45.2-APC, B220-APC-Cy7, F4/80-PE, F4/80-PErCP-Cy5.5, IgD-PE, and TNF-α–PE antibodies were from BioLegend. IgM-efluor650, CD4-efluor650, and CD11b-efluor605 antibodies were from eBioscience.
Shen L., Chng M., Alonso M.N., Yuan R., Winer D.A, & Engleman E.G. (2014). B-1a Lymphocytes Attenuate Insulin Resistance. Diabetes, 64(2), 593-603.
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8

Mice Tail Bleed and Blood Panel Staining

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Mice were tail bled into a 150-µl Alsever’s solution. Samples where then treated with 10 ml Ammonium-Chloride-Potassium (ACK) Lysing Buffer for 2 min and centrifuged (1600 rpm) for 5 min. The supernatant was removed, washed in 5 ml SM, centrifuged again, and finally plated in a 96-U well for 1–3 h for staining. Blood panel staining: CD45.1-Apc, CD45.2-PacBlue, Mac1-PeCy7, B220-ApcCy7, and CD3e-PE (all from Biolegend, San Diego, CA).
Keinan N., Scharff Y., Goldstein O., Chamo M., Ilic S, & Gazit R. (2021). Syngeneic leukemia models using lentiviral transgenics. Cell Death & Disease, 12(2), 193.
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9

Immunostaining and Flow Cytometry Protocols

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The following Abs were used for IF and IHC staining: anti-mouse IgA Ab-PE (Abcam, 97013) or fluorescein isothiocyanate (FITC; Abcam, 97234), anti-mouse CD138-PE (clone 281-2, BioLegend), anti-human IgA (DAKO, #IR51061), or CD138 (DAKO, M7228). For flow cytometry, the following Abs against mouse antigens were used: B220–APC-Cy7 (clone RA3-6B2, BioLegend), CD138-PE or BV421 (clone 281-2), GL7-PerCP Cy5.5 (clone GL7, BioLegend), Ki67–PE-Cy7 (clone B56, BD), IgG1-FITC (clone A85-1, BD), IgG2a/c-FITC (clone RMG2a-62, BioLegend), IgG2b-FITC (SouthernBiotech, 1092-02), IgG3-FITC (SouthernBiotech, 1102-02), IgA-biotin (clone RMA-1, BioLegend), CD73-biotin (clone TY/11.8, BioLegend), CD140b-APC (clone APB5, BioLegend), CD105–PE-Cy7 (clone MJ7/18, BioLegend), CD45–PerCP-Cy5.5 (clone 30-F11, BioLegend), and CD31-PE (clone 390, BioLegend); other reagents were also used: rabbit polyclonal anti-SPTBN1 (amino acids 2100 to 2150; Abcam, 72239), normal rabbit IgG (an isotype-matched control for the latter; Sigma-Aldrich, 12-370), anti-rabbit IgG-BV421 (clone Poly4064, BioLegend), streptavidin-BV421 (BioLegend, 405226), or APC (BioLegend, 405207).
Nihei Y., Haniuda K., Higashiyama M., Asami S., Iwasaki H., Fukao Y., Nakayama M., Suzuki H., Kikkawa M., Kazuno S., Miura Y., Suzuki Y, & Kitamura D. (2023). Identification of IgA autoantibodies targeting mesangial cells redefines the pathogenesis of IgA nephropathy. Science Advances, 9(12), eadd6734.
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10

Comprehensive Immune Cell Profiling

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Antibodies used: CD45.1-Apc, CD45.2- PacBlue, Ter119-PerCPCy5.5, Mac1-PeCy7, B220-ApcCy7, CD3e-PE, cKit-ApcCy7, Sca1-APC, CD150-PeCy7, CD48--PerCPCy5.5, Gr1-PacBlue, and CD19-APC (all from Biolegend, San Diego, CA). Cells were stained on ice for 30 min, washed, filtered through a 70-µm filter, and analyzed using Fluorescence-Activated Cell Sort machine (FACS). Routine analysis was performed by using 4-laser Gallios (Beckman Coulter) and the Kaluza analysis software (Version 2.1). Sorting used of a 6-laser, BD FACSAria™ III sorter.
Keinan N., Scharff Y., Goldstein O., Chamo M., Ilic S, & Gazit R. (2021). Syngeneic leukemia models using lentiviral transgenics. Cell Death & Disease, 12(2), 193.
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