B220 biotin

Manufactured by Thermo Fisher Scientific

Sourced in United States

The B220-biotin is a laboratory instrument designed for biomolecular analysis. It is a key component used in various research and diagnostic applications. The core function of the B220-biotin is to enable the detection and quantification of biotinylated molecules, such as proteins, nucleic acids, and other biomolecules. The device provides a reliable and efficient means to conduct these types of analyses within a laboratory setting.

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4 protocols using b220 biotin

Multicolor Flow Cytometry Analysis of Immune Cells

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Cell suspensions from SPL, mLN, and cLP were analyzed by flow cytometry. Conventional CD4⁺ T cells and Tregs were stained with anti-CD3ε and anti-CD4 mAbs (BD Biosciences, San Jose, CA) and intracytoplasmic anti-foxp3 mAbs (eBioscience, San Diego, CA). Neutrophils were stained with Gr-1-FITC, CD11b-PECy7, and Ly6G-AF647 mAbs (eBioscience). Plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) were stained with MHCII-FITC, CD11b-PECy7, PDCA-1-AF647, and CD11c-APCCy7 mAbs with a lineage-negative mAb cocktail containing B220-biotin, CD3ε-biotin, Ly6G-biotin (eBioscience) and PerCP-Cy5.5-conjugated streptavidin. The cDC subsets were stained with MHCII-FITC, CD11c-APCCy7, CD11b-PECy7, and CD103-PE mAbs with a lineage-negative mAb cocktail containing B220-biotin, CD3ε-biotin, Ly6G-biotin (eBioscience) and PerCP-Cy5.5-conjugated streptavidin (eBioscience). Monocytes and macrophages (MΦ) were stained with Gr-1-FITC, MHCII-PE, Ly6C-PerCP-Cy5.5, CD11b-PECy7, Ly6G-APC, and CD11c-APCCy7 mAbs (eBioscience). Blocking of FcγR binding was performed using mouse and rat serum (Jackson ImmunoResearch, West Grove, PA). Cells were analyzed on a FACS Canto II (BD Biosciences). Data were collected using FACS Diva software (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR).

Wurbel M.A., Bras S.L., Ibourk M., Pardo M., McIntire M.G., Coco D., Geha R.S., Fiebiger E, & Snapper S.B. (2014). CCL25/CCR9 Interactions Are Not Essential for Colitis Development but Are Required for Innate Immune Cell Protection from Chronic Experimental Murine Colitis. Inflammatory bowel diseases, 20(7), 1165-1176.

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Isolation and Characterization of Murine Early B Cells

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BM cells were stained with biotinylated antibody B220 Biotin (eBioscience, 13–0452–82) following initial Fc-block incubation. After incubation, cells were washed twice with PBS/5% FCS, centrifuged and incubated with Anti-Biotin MicroBeads (Miltenyi Biotec, 130–090–485). Magnetic purification was performed according to the manufacturer’s instructions. B220+ cells were counted and stained with SAv-Brilliant Violet 605™ (BioLegend, 405229) and anti-Mouse IgM PE-Cyanine7 Antibody (Life Technologies, 25–5790–81). After wash, cells were stained with 7-Aminoactinomycin D (7AAD; Biotium 40037) and phenotypically defined early B precursors (7AAD⁻B220⁺IgM⁻) sorted using a BD FACSAria II cell sorter (BD Biosciences). Analysis was performed using Flowjo software version 10.3 (Flowjo LLC, OR, USA).

Valletta S., Thomas A., Meng Y., Ren X., Drissen R., Sengül H., Di Genua C, & Nerlov C. (2020). Micro-environmental sensing by bone marrow stroma identifies IL-6 and TGFβ1 as regulators of hematopoietic ageing. Nature Communications, 11, 4075.

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Murine Model of Experimental Autoimmune Encephalomyelitis

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Myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) peptide (MEVGWYRSPFSRVVHLYRNGK) was generated by the Protein Core laboratory of the Blood Research Institute, BloodCenter of Wisconsin. The 2.4G2 hybridoma was obtained from the American Tissue Culture Collection. Anti-mouse CD4-APC-eFluor 780, CD25-Alexa Fluor 700, CD11b-PE, CD11b-biotin, IL-17-Alexa Fluor 647, CD11c-Biotin, B220-Biotin, CD8-Biotin, CD11b-biotin, Foxp3-PE and streptavidin-PE Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-mouse B220-PE-Texas Red, IFN-γ-PE, anti-active caspase 3-FITC and anti-human Ki67-FITC were purchased from BD Biosciences (San Diego, CA). Anti-mouse Ly-6C-APC and Ly-6G-APC-Cy7 were purchased from Biolegend (San Diego, CA). Anti-mouse/rat Neuropilin-1-APC was obtained from R&D Systems (Minneapolis, MN). Monoclonal antibodies SMI-32 (anti-nonphosphorylated neurofilament-H) and SMI-99 (anti-myelin basic protein (MBP)) were purchased from Covance (Emeryville, CA). Strepavidin Alexa 405 and goat anti-mouse Alexa 546 (H + L) were purchased from Life Sciences Advanced Technologies (St. Petersburg, FL). Anti-Biotin microbeads were purchased from MiltenyiBiotec (Auburn, CA).

Ray A., Basu S., Miller N.M., Chan A.M, & Dittel B.N. (2014). An Increase in Tolerogenic Dendritic Cell and Natural T Regulatory Cell Numbers During EAE in Rras−/− Mice Results in Attenuated Disease. Journal of immunology (Baltimore, Md. : 1950), 192(11), 5109-5117.

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Flow Cytometry Antibody Staining Protocol

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For the flow cytometry analyses, cells were stained with monoclonal antibodies against the following anti-mouse molecules: CD3-biotin, Ter119-biotin, GR-1-biotin, B220-biotin, CD11b-biotin, NK1.1-biotin, CD3-biotin, CD4-biotin, CD8-biotin, cKit-BV650, Sca1-PE, CD135-PerCP, CD34-APC, CD25-PE, CD44-APC, CD3-APC, CD8-PerCP, CD-BV650, CD45.1/Ly5.1-PE-Cy7, CD45.2/Ly5.2-APC-Cy7 (all from eBiosciences, CA, USA). For secondary detection of biotinylated antibodies, streptavidin conjugated with FITC or PE-Cy7 was used (eBiosciences, San Diego, CA, USA). All flow cytometry measurements (Canto II BD biosciences or LSRII BS Biosciences) were calibrated first with BD™ CompBead Plus, κ/Negative Control (BSA) Compensation Plus (7.5 µm). Recommended flow cytometry settings are specified in [17 ]. Flow cytometry analyses were performed using FlowJo software (Treestar, Ashland, OR, USA).

de Roo J.J., Chhatta A., Garcia-Perez L., Naber B.A., Vloemans S.A., Salvatori D.C., Pike-Overzet K., Mikkers H, & Staal F.J. (2022). Axin2/Conductin Is Required for Normal Haematopoiesis and T Lymphopoiesis. Cells, 11(17), 2679.

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