B220 percp cy5

Manufactured by BioLegend

B220 PerCP-Cy5.5 is a fluorescent-conjugated antibody that binds to the B220 antigen, also known as CD45R. It is commonly used in flow cytometry applications to identify and analyze B cells in biological samples.

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PerCP-Cy5.5 (133 citations) B220-PerCP (6 citations) Anti-B220-PerCP/Cy5.5 (5 citations) B220-PerCP-Cy5.5 (RA3-6B2, (2 citations) PerCP-Cy5.5-anti-B220 (2 citations) PerCP/Cy5.5‐B220 (2 citations)

5 protocols using b220 percp cy5

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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Multiparametric Flow Cytometry Analysis of T Cell Subsets

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CD1a-PE, CD1b-PE, CD1c-FITC, B220-PerCPCy5.5, F4/80-APC, CD11c- BV421, CD8-BV510, CD4-PerCPCy5.5, IFN-γ-APC, IL-17-PE, Granzyme B-BV421, and TNF-α-FITC antibodies were purchased from BioLegend (San Diego, CA). TCR-β-BV421, anti-Vβ3 and 4-PE, and anti-Vβ5.1/5.2-APC were obtained from BD biosciences (San Jose, CA). For flow cytometry, single cell suspensions from organs were incubated with 2.4G2 FcR blocking antibody for 10 minutes and then stained in HBSS-2% FBS containing 50 μg/mL gentamicin. Samples were run on the FACS Canto II flow cytometer (BD Biosciences, San Jose, CA). Analysis of flow cytometry data was performed on FlowJo software (Tree Star, Inc). S12 Fig shows the gating strategies employed for the FACS plots of group 1 CD1-restricted T cells in the polyclonal setting in Fig 3 and group 1 CD1-restricted T cell line functional assays in Fig 5.

Visvabharathy L., Genardi S., Cao L., He Y., Alonzo F I.I.I., Berdyshev E, & Wang C.R. (2020). Group 1 CD1-restricted T cells contribute to control of systemic Staphylococcus aureus infection. PLoS Pathogens, 16(4), e1008443.

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Multicolor Flow Cytometry Analysis of Immune Cells

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The following antibodies were used to stain PCW cells: B220 PerCP-Cy5.5 (BioLegend, 103236), CD5 PE-Cy7 (BioLegend, 100622), CD19 BUV395 (BD Biosciences, 563557), IgM APC (Biolegend, 406509), PD-L2 BV421 (BD Biosciences, 564245), CD11b APC-Cy7 (BioLegend, 101226), Live/dead Ghost violet 510 (Tonbo Biosciences, 13-0870). For cells isolated from PZTD^+/+ mice, ZsGreen fluorescent protein was analyzed using the same channel as FITC. For cells isolated from CD19-Cre^+/- PTZD^+/+ mice, TdTomato fluorescent protein was analyzed using the same setting for TexasRed. To analyze TILs, CD45 PerCP-Cy5.5 (eBioscience, 45-0451-80), was added to the above panel replacing B220 for leukocyte gating. The stained samples were analyzed at the Boston University Medical Campus Flow Cytometry core facility using a BD LSRII flow cytometer and a Beckman Coulter MoFlo for cell sorting. Live leukocytes were gated by forward and side scatters, live/dead Ghost violet 510 and CD45 staining. B1 cells were gated as B220IntCD5Int population from the live leukocyte gate. L2pB1 cells were gated as PD-L2+IgM+ from the B1 cell gate. In the transgenic-knock in mice, L2pB1 cells were also identified by the expression of ZsGreen and TdTomato (Supplementary Figure S1).

Shibad V., Bootwala A., Mao C., Bader H., Vo H., Landesman-Bollag E., Guo C., Rubio A., Near R., Gao W., Challa S., Chukka V., Gao J., Kelly A., Landesman T., VanHelene T, & Zhong X. (2021). L2pB1 Cells Contribute to Tumor Growth Inhibition. Frontiers in Immunology, 12, 722451.

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Comprehensive B Cell and Immune Profiling

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Flow-cytometric analysis or cell sorting of single cell suspensions generated from spleen or bone marrow was performed on an LSR-Fortessa or a FACS-Aria-III, respectively (both BD) and analyzed using FlowJo v10 software. Antibodies used (40 µl/sample): B220-FITC (biolegend RA3-6B2) 1:300, B220-APC/eF780 (ebioscience RA3-6B2) 1:200, B220-PE (BD RA3-6B2) 1:300, B220-PErCP-Cy5.5 (biolegend RA3-6B2) 1:300, CD19-BV605 (biolegend 6D5) 1:400, IgM-APC (biolegend RMM-1) 1:300, IgM-FITC (BD II/41) 1:300, IgM-eF450 (ebioscience eb121-15F9) 1:200, IgD-PerCP-Cy5.5 (biolegend 11-26c2a) 1:400, TCRβ-BV605 (BD H57-597) 1:200, TCRβ-FITC (ebioscience H57-597) 1:300, CD4-APC/Cy7 (BD GK1.5) 1:400, cKit-PE/Cy7 (biolegend 2B8) 1:300, cKit-PerCP-Cy5.5 (biolegend 2B8) 1:300, cKit-BV421 (biolegend 2B8) 1:300, Mac1-APC (ebioscience M1/70) 1:300, CD25-PE (biolegend PC61) 1:500, CD93-PE/Cy7 (ebioscience AA4.1) 1:300, NK1.1-APC (biolegend PK136) 1:300, AnnexinV-FITC (biolegend Lot: B206041) 1:1800, AnnexinV-eF450 (ebioscience; Lot: E11738-1633) 1:1000. FITC-F(ab’)₂ Fragment Goat Anti-Mouse IgM [1 μg/ml], μ Chain Specific (Jackson ImmunoResearch).

Schuler F., Weiss J.G., Lindner S.E., Lohmüller M., Herzog S., Spiegl S.F., Menke P., Geley S., Labi V, & Villunger A. (2017). Checkpoint kinase 1 is essential for normal B cell development and lymphomagenesis. Nature Communications, 8, 1697.

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Immunophenotyping B and T Cells

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WT and mFICD^−/− mice aged 6–8 weeks were euthanized by CO₂ asphyxiation followed by cervical dislocation. Spleen, bone marrow, and thymus tissues were extracted and homogenized in PBE buffer (PBS +0.5% BSA and 1 mM EDTA). Red blood cells were lysed using ACK lysis buffer (Gibco), and cells were resuspended in PBE for surface staining with the following antibodies (clone;source) for 30 min at 4˚C: B220 PerCP-Cy5.5, CD43 APC, CD19 Pacific Blue, IgM FITC, IgD APC-Cy7, CD21 PerCP-Cy5.5, CD23 FITC, IgM APC, CD44 FITC, CD25 PE-Cy7, CD4 APC, CD8α Pacific Blue (53–6.7;BioLegend), CD3 PerCP-Cy5.5 (145-2C11;BD), B220 APC-Cy7. All samples were blocked using Fc-Block (BD Bioscience). Acquisition of B and T cell populations was performed on an LSRFortessa cytometer (BD) instrument and analyzed with the FlowJo software package (Tree-Star).

McCaul N., Porter C.M., Becker A., Tang C.H., Wijne C., Chatterjee B., Bousbaine D., Bilate A., Hu C.C., Ploegh H, & Truttmann M.C. (2021). Deletion of mFICD AMPylase alters cytokine secretion and affects visual short-term learning in vivo. The Journal of Biological Chemistry, 297(3), 100991.

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