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B220 pe

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B220-PE is a fluorescently conjugated antibody that binds to the B220 (CD45R) antigen. B220 is a high molecular weight isoform of the CD45 protein expressed on the surface of B cells. The PE (phycoerythrin) fluorophore is used to label the antibody, enabling detection and analysis of B cells by flow cytometry.

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Close spelling variants of this product

Anti-B220-PE (9 citations) B220 PE-Cy7 (6 citations) Anti-B220-PE-Cy7 (4 citations) B220 (PE RA3-6B2; (4 citations) Anti-B220-PE (RA3-6B2, (3 citations) CD45R/B220-PE-Cy7 (3 citations) Anti-mouse B220 Pe-Cy7 (2 citations) PE-conjugated anti-B220 (2 citations) PE-conjugated anti-mouse B220 antibody (2 citations) PE-conjugated rat anti-mouse B220 (2 citations)

15 protocols using b220 pe

1

Multicolor Flow Cytometry Panel

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Abs against following antigens: CD3-FITC, CD3-APC, CD25-FITC, CD4-PE, CD4-PECy7, CD8-PE, B220-PE, NK-1.1-PE, CD69-APC, FOXP3-PE, IFN-γ-APC, Isotype-matched control Ab, Annexin V-FITC, 7AAD, Purified anti-CD25 mAb (PC61) were purchased from BioLegend (San Diego, CA). The murine recombinant cytokines (IL-2, IL-12) were from Peprotech (Rocky Hill, NJ).
Huo R., Wang L., Wang X., Zhao Y., Wang Y., Zhao X., Chang L., Liu S.L., Tong D., Zhang H, & Huang Y. (2016). Removal of regulatory T cells prevents secondary chronic infection but increases the mortality of subsequent sub-acute infection in sepsis mice. Oncotarget, 7(10), 10962-10975.
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2

Multiparametric Flow Cytometry of Immune Cells

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BAL cells were stained separately with two antibody cocktails. The first was for NK cells with NK1.1-PE (BD PharMingen), T cells with TCRβ-APC (BD PharMingen) and NKT cells NK1.1-PE and TCRβ-APC. The second stain was for neutrophils with Ly6G-FITC (BD PharMingen) and B cells with B220-PE (Biolegend). NKT cells were identified using CD1dα-GALCER tetramer conjugated to APC. Propidium Iodide was used to exclude dead cells before data were acquired using either FACSCalibur or FACSCantoII.
Bird N.L., Olson M.R., Hurt A.C., Oshansky C.M., Oh D.Y., Reading P.C., Chua B.Y., Sun Y., Tang L., Handel A., Jackson D.C., Turner S.J., Thomas P.G, & Kedzierska K. (2015). Oseltamivir Prophylaxis Reduces Inflammation and Facilitates Establishment of Cross-Strain Protective T Cell Memory to Influenza Viruses. PLoS ONE, 10(6), e0129768.
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3

Isolation and Stimulation of Splenic B Cells

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Splenic B cells of TAPtag mice were isolated using CD43 magnetic beads (Miltenyi Biotec) negative selection and cultured in RPMI1640 containing 15% FBS with 20 μg/ml LPS (Sigma-Aldrich) and/or 20 ng/ml IL4 (R&D) for 48~72 hrs. CH12F3 cells were cultured in RPMI1640 containing 10% FBS and stimulated with 20 μg/ml LPS (Sigma-Aldrich), 20 ng/ml IL4 (R&D) and 1 ng/ml TGFβ (R&D). Stimulated splenic B cells were stained with B220-PE (Biolegend) and IgG1-FITC (BD Bioscience) and stimulated mutant CH12F3 cells were stained with IgA-FITC (BD Bioscience). Cells were analyzed by using LSRII and FlowJo (BD Bioscience).
Lim J., Giri P.K., Kazadi D., Laffleur B., Zhang W., Grinstein V., Pefanis E., Brown L.M., Ladewig E., Martin O., Chen Y., Rabadan R., Boyer F., Rothschild G., Cogné M., Pinaud E., Deng H, & Basu U. (2017). Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry. Cell, 169(3), 523-537.e15.
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4

Multiparameter Flow Cytometry Analysis

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FACS analysis were performed with routine protocols using the FACS Calibur flow cytometer (BD Immunocytometry Systems), antibodies used for mice spleen lymphocyte staining are as following: CD3-Percp cy5.5, CD4-FITC, APC, CD8-Percp cy5.5, PE, CD19-FITC, B220-PE, CD69-FITC, CD80-FITC, CD86-APC, CD40-PE (Biolegend) and bone marrow staining: F4/80-PE, CD11b-FITC, MHC-II-PerCP cy5.5 (Biolegend). FACS buffer; 1 × PBS (Gibco) + 2% FBS (Sigma). All data were analyzed using FlowJo (Tree Star, Inc.).
Ying Q., Ma T., Cheng L., Zhang X., Truax A.D., Ma R., Liu Z., Lei Y., Zhang L., Ye W., Zhang F., Xu Z., Shang L., Liu R., Wang F, & Wu X. (2016). Construction and immunological characterization of CD40L or GM-CSF incorporated Hantaan virus like particle. Oncotarget, 7(39), 63488-63503.
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5

Multiparameter Flow Cytometry Analysis

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Spleens were mechanically disrupted, single-cell suspensions were generated, and red blood cells were lysed with ammonium chloride TRIS. Cells were resuspended in PBS containing 1% FBS and incubated with indicated antibodies. For analysis of cell surface markers, antibodies against the following molecules were used: B220-PE (RA3–6B2; BioLegend), B220-BV786 (RA3–6B2; BD Biosciences), B220-BV510 (RA3–6B2; BioLegend), CD4-BV711 (GK1.5; BioLegend), CD8 BV421 (53–6.7; BioLegend), CD138-PECy7 (281–2; BioLegend), CD69-BV786 (H1.2F3; BD Biosciences), CD86-PerCPCy5.5 (GL-1; BioLegend). After cell surface staining, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD) (as per manufacturer’s instructions) and stained with Dylight650-E4 anti-Ars/A1 idiotype (produced and conjugated in our laboratory) OR HEL-650 (produced and conjugated in our laboratory) and Alexa Fluor 488-anti-GFP (rabbit polyclonal; Life Technologies). Events were collected on a CyAn ADP (Dako) and subsequent analysis using FlowJo software (Tree Star).
Franks S.E., Getahun A, & Cambier J.C. (2019). A Precision B cell-targeted Therapeutic Approach to Autoimmunity Caused by PI3K Pathway Dysregulation. Journal of immunology (Baltimore, Md. : 1950), 202(12), 3381-3393.
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6

Multiparametric Flow Cytometry of Murine and Human B-Cells

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Single‐cell suspensions were stained with a mixture of 5 to 6 fluorophores including and 7‐aminoactinomycin D (7‐AAD) for viability and acquired on the BD Verse flow cytometer with data analyzed using FlowJo software (BD Biosciences). Human B‐cell subsets were identified using the following antibodies: CD19‐APC, CD27‐BV510, CD38‐APC‐Cy7, CD24‐BV421, and IL‐10‐PE‐Cy7 (all from BioLegend). Mouse B‐cell subsets were identified using the following antibodies: B220‐PE, CD23‐APC, CD21‐PE‐Cy7, IgM‐BV421, CD138‐APC, CD86‐PE‐Cy7, CD69‐APC‐Cy7, and IL‐10‐APC‐Cy7 (all from BioLegend). Intracellular IL‐10 expression was detected as previously reported.33 Immunophenotype of human and murine B‐cell subsets analyzed are listed in Supplementary Table 1.
Lee W., Wang L., Yen M., Hsu P., Lee Y., Liu K., Lin K., Su Y., Sytwu H, & Yen B.L. (2021). Resident vs nonresident multipotent mesenchymal stromal cell interactions with B lymphocytes result in disparate outcomes. Stem Cells Translational Medicine, 10(5), 711-724.
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7

Tissue Dissociation and Cell Isolation

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Splenocytes were isolated by mincing and mechanical dissociation of tissue through a 40-mm cell strainer. Cells were isolated into phosphate-buffered saline with 1% bovine serum albumin, 2 mM EDTA, and 25 mM HEPES. Red cells were removed by ammonium-chloride-potassium lysis. Cells were then stained with either a myeloid marker panel (CD45-BV421, CD11b-BV605, Ly6C-FITC, Ly6G-PECy7; BioLegend) or lymphoid marker panel (CD45-BV421, CD11b-BV605, B220-PE, CD3e-FITC; BioLegend). Cells were analyzed on an Attune cytometer and reported as the percentage of CD45 cells.
Hill A., Khalil H., Laborc K., Kounelis-Wuillaume S., Gavade S., Johnston C., Singer B.H, & Spencer-Segal J.L. (2023). Corticosteroid Treatment During Sepsis Alters Hippocampal Function in Male and Female Survivors. Biological Psychiatry Global Open Science, 4(1), 336-345.
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8

Fetal Liver and Spleen Analysis

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The fetal livers and spleens of transplanted embryos (E18.5) were isolated and homogenized using 18 G needles with 1 ml syringes or microscope slides (Matsunami Glass Ind., Ltd., Osaka, Japan). After ACK lysis buffer treatment, cells were stained with the following antibodies. For mouse HSC-transplanted embryos, anti-mouse CD45.1 APC or PE, CD45.2 PE/Cy7 or FITC, CD11b APC/cy7, B220 PE, and CD3 PE (BioLegend, San Diego, CA) were used. For rat HSC-transplanted embryos, anti-mouse CD45.2 FITC or PerCP and anti-rat CD45 PE (BioLegend) were used. Erythroid cells were detected from the fetal livers of rat HSC-transplanted embryos by staining with anti-mouse and anti-rat erythroid cell antibodies (BioLegend). Gallios, CytoFLEX (Beckman Coulter, Brea, CA) and Kaluza v1.3, CytExpert software (Beckman Coulter) as well as BD-LSR (BD Biosciences, San Jose, CA) and FlowJo software (TreeStar, Ashland, OR) were used for the data acquisition and analysis.
Jeon H., Asano K., Wakimoto A., Kulathunga K., Tran M.T., Nakamura M., Yokomizo T., Hamada M, & Takahashi S. (2021). Generation of reconstituted hemato-lymphoid murine embryos by placental transplantation into embryos lacking HSCs. Scientific Reports, 11, 4374.
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9

Isolation and Characterization of Dendritic Cells

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Freshly isolated live (7AAD2; BD Biosciences) dLN cells and in vitro– derived BM cells were stained with combinations of fluorochrome-conjugated Abs to CD4–Pacific Blue or CD4-PE/Cy5 (clone GK 1.5); CD3-PE/Cy7, CD3-PE/Cy5, CD3-PE, or CD3-FITC (17A2); CD11c-PE/Cy7, CD11c-FITC, or CD11c-Orange 605 (N418); CD11b-PE/Cy7 or CD11b-FITC (M1/70); B220-PE or B220-BV 510 (RA3-6B2); CCR7-PE (4B12); Siglec-H–Pacific Blue or Siglec-H–FITC (551); PDCA-1–PE or PDCA-1–FITC (927); CD19-PE/Cy7 or CD19-PE/Cy5 (6D5); CD25-PE (PC61.5) (BioLegend); and T1ST2 (DIH9) (T1ST2-FITC [MD Biosciences] or T1ST2–PE [BioLegend]). For intranuclear staining of Foxp3, a permeabilization kit and Abs (Foxp3–Pacific Blue or Foxp3-PE/CyC5; clone FJK-16s) were used (eBioscience). Flow cytometric measurements were performed using an Attune Acoustic Focusing Cytometer (Applied Biosystems) and a FACSAria III (BD). FACS sorting of pDCs was performed using a FACSAria III. Data analysis was performed with FlowJo software (TreeStar).
Alissafi T., Kourepini E., Simoes D.C., Paschalidis N., Aggelakopoulou M., Sparwasser T., Boon L., Hammad H., Lambrecht B.N, & Panoutsakopoulou V. (2018). Osteopontin Promotes Protective Antigenic Tolerance against Experimental Allergic Airway Disease. Journal of immunology (Baltimore, Md. : 1950), 200(4), 1270-1282.
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10

Induction of Ig Isotypes in B Cells

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CD43-negative B cells from spleen were stimulated with LPS (20 μg/ml) (Sigma-Aldrich) for IgG3; LPS (20 μg/ml) and IL4 (20 ng/ml) (PeproTech) for IgG1; and LPS (10 μg/ml) (Sigma-Aldrich), TGF-β (2 ng/ml) (R&D Systems), and anti-IgD dextran (0.33 μg/ml) (Fina Biosolutions) for IgG2b and IgA CSR. CSR efficiency of each isotype was measured after 3 to 4 days. B220-PE (BioLegend)/IgG3-FITC (fluorescein isothiocyanate) (BD Biosciences) and B220-FITC (BD Biosciences)/IgG2b-PE (BioLegend), IgA-PE (eBioscience), or IgG1-PE (BD Biosciences) were used for staining cells for FACS analyses.
Rothschild G., Zhang W., Lim J., Giri P.K., Laffleur B., Chen Y., Fang M., Chen Y., Nair L., Liu Z.P., Deng H., Hammarström L., Wang J, & Basu U. (2020). Noncoding RNA transcription alters chromosomal topology to promote isotype-specific class switch recombination. Science immunology, 5(44), eaay5864.
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