B220 fitc

Manufactured by Thermo Fisher Scientific

Sourced in United States

The B220-FITC is a fluorescently-labeled antibody that binds to the B220 antigen, also known as CD45R, which is expressed on the surface of B lymphocytes. It can be used for the identification and enumeration of B cells in flow cytometry applications.

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11 protocols using b220 fitc

Regulatory T cell Immunophenotyping

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Peripheral blood mononuclear cells (PBMCs) were stained with the following antibodies, B220-FITC, CD4-e450, CD8-BV510, CD25-BV605, and FoxP3-e660 using the FoxP3 staining buffer kit from eBioscience per manufacturer's instructions. Data were collected on an Attune NXT flow cytometer and cell population analysis was conducted using FCS Express 7 (De Novo software, Glendale, CA).

Biswas M., Palaschak B., Kumar S.R., Rana J, & Markusic D.M. (2020). B Cell Depletion Eliminates FVIII Memory B Cells and Enhances AAV8-coF8 Immune Tolerance Induction When Combined With Rapamycin. Frontiers in Immunology, 11, 1293.

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Comprehensive Hematopoietic Cell Analysis Protocol

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Flow assisted cell sorting (FACS) was performed as described elsewhere (16 (link)) with the following conjugated antibodies; Mac1 PE, Gr1 FITC, B220 APC, B220 FITC, CD19 PE, surface IgM PE, CD43 FITC, CD3 PE, CD4 APC, CD8 FITC, CD71 PE, Ter119 FITC, Sca-1 PE, cKit FITC, cKit APC-Cy7, IL7Ra PECy7, CD34 FITC, CD16/32 PE (eBiosciences or BD Biosciences). StemSep Mouse Hematopoietic Progenitor Kit Lineage Positive antibody cocktail (Stem Cell Technologies, Vancouver, Canada) was used to detect lineage positive bone marrow cells, stained as previously described (16 (link)). Apoptosis assays were conducted using AnnexinV-FITC Apoptosis Detection Kit I following the manufacturer’s recommended protocol. IHC and immunoblotting were performed as previously described (49 (link)). Formalin-fixed paraffin embedded sections were stained with hematoxylin and eosin (H&E), myeloperoxidase (MPO) (A0398, DAKO), CD3 (MCA1477, AbD Serotec), B220 (553086, BD), Ter119 (116201, BioLegend). Stained slides were scanned and imaged as described elsewhere (49 (link)). For immunoblots, primary antibodies used were anti-V5 (ab9116, Abcam or R960-25, Life Technologies), and beta-actin (Cell Signaling Technology). Protein was visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific, Illinois, USA) and Amersham Hyperfilm ECL (GE Healthcare Ltd, Buckinghamshire, UK).

Gough S.M., Lee F., Yang F., Walker R.L., Zhu Y.J., Pineda M., Onozawa M., Chung Y.J., Bilke S., Wagner E.K., Denu J.M., Ning Y., Xu B., Wang G.G., Meltzer P.S, & Aplan P.D. (2014). NUP98-PHF23 is a chromatin modifying oncoprotein that causes a wide array of leukemias sensitive to inhibition of PHD domain histone reader function. Cancer discovery, 4(5), 564-577.

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Comprehensive Immune Cell Profiling

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Lymphocytes from each organ was stained with various combinations of mAbs to CD4-FITC/APC, CD8α-PE/APC, CD3α-PE, and B220-FITC (eBiosciences), CD103-APC, LAP (TGF-β1)-PE (Clone TW7-20B9, Biolegend). Fixation/Permeabilization Concentrate and Diluent Kit (eBiosciences) were used for the intracellular staining of FoxP3-PE/APC according to the user’s handbook. After stained with surface markers, cells were stained with Annexin V and PI (BD Pharmingen) to examine apoptosis. For in vivo BrdU incorporation assay, mice were administered of BrdU (16 mg/ml; 50 μl) via i.n. route [48 (link)]; cells were harvested after 24hours and stained with BD FastImmune BrdU kit (BD Biosciences). Samples were run on BD Biosciences FACSCalibur, FACSAria or Accuri C6 instruments, and analyzed with FlowJo software (Tree Star, Ashland, OR).

Li C., Jiao S., Wang G., Gao Y., Liu C., He X., Zhang C., Xiao J., Li W., Zhang G., Wei B., Chen H, & Wang H. (2015). The Immune Adaptor ADAP Regulates Reciprocal TGF-β1-Integrin Crosstalk to Protect from Influenza Virus Infection. PLoS Pathogens, 11(4), e1004824.

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Tumor Dissociation and Immune Cell Analysis

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Tumors were minced into thin pieces and were dissociated in DNase I (1 U/ml; Roche, Indianapolis, IN, USA), collagenase D (1 mg/ml; Roche), and 0.125% trypsin-EDTA (Gibco) in pre-warmed DMEM (Welgene) for an hour at 37 °C with gentle agitation. The tissues were mechanically dissociated on a 100 μm nylon mesh strainer. Next, the single cells were passed through a 40 μm nylon mesh strainer. Spleens were also mechanically dissociated on 40 μm nylon mesh strainer. RBCs were lysed for 5 minutes in 1X Pharmlyse buffer.
Cells were stained with fluorescently tagged antibodies. All data were detected by a FACSCalibur cytometric system and analyzed by FlowJo software. The following fluorophore-labeled antibodies were purchased from e-bioscience (San Diego, CA, USA): CD45-FITC, CD3-FITC, CD4-PE, CD4-FITC, CD8-APC, CD11b-APC, CD11c-PE, CD25-PE, CD25-APC, B220-FITC, CD19-PE, CD86-APC. Foxp3-Alexa Fluor647 was purchased from BD bioscience, and F4/80-PE and CD206-APC were obtained from Biolegend (San Diego, CA, USA).

Lee C., Bae S.J., Joo H, & Bae H. (2017). Melittin suppresses tumor progression by regulating tumor-associated macrophages in a Lewis lung carcinoma mouse model. Oncotarget, 8(33), 54951-54965.

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Phenotypic Identification of Migratory DCs

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Freshly isolated mesenteric lymph nodes (MLNs) were analyzed by flow cytometry (14 (link)). Identification of migratory DCs in the MLNs was performed by gating CD103+ cells out of CD11c+ MHC-II cells in the MLNs (the gating strategy is shown in Figure 2A). Cells obtained and resuspended in PBS with 1% bovine serum albumin were incubated with antimouse CD16/CD32 (Mouse BD Fc Block; BD Pharmingen) for 20 min on ice to block nonspecific binding sites. For surface staining, cells were incubated with CD4-PerCp-Cy5.5, CD69-PE, CD25-AlexaFluor488, CD11c-PerCp-Cy5.5, CD103-APC, CD40-FITC, CD86-PE-cy7, MHCII-PE, CD3-Percy5.5, CD27-PE, CD19-APC, B220-FITC (eBiosciences). Foxp3-PE-cy7, and Tbet-APC (eBioscience) were used for intracellular staining. Staining and flow cytometry were performed as described previously (14 (link)).

Xiao L., Engen P.A., Leusink-Muis T., van Ark I., Stahl B., Overbeek S.A., Garssen J., Naqib A., Green S.J., Keshavarzian A., Folkerts G, & van't Land B. (2019). The Combination of 2′-Fucosyllactose with Short-Chain Galacto-Oligosaccharides and Long-Chain Fructo-Oligosaccharides that Enhance Influenza Vaccine Responses Is Associated with Mucosal Immune Regulation in Mice. The Journal of Nutrition, 149(5), 856-869.

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Peritoneal Cell Harvesting and Characterization

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To harvest peritoneal cells, 5 mL of Dulbecco’s Modified Eagle Medium (DMEM) containing 5% fetal bovine serum (FBS) was injected into the peritoneum of mice using a 27G needle. After injection, the DMEM was collected from the peritoneum using a 5ml syringe with a 25G needle. RBCs were lysed using ACK Lysing Buffer (Quality Biological). Peritoneal Cells were resuspended at 10⁶ cells/ml, blocked with Fc blocker and stained for the lineage markers CD19-Percp (Biolegend), B220-FITC (eBioscience), CD11c-PECy7 (Biolegend), B220-APC (eBioscience), CD3-FITC (BD Pharmingen), CD11b-PE (Biolegend).

Corradetti C., Jog N.R., Gallucci S., Madaio M., Balachandran S, & Caricchio R. (2016). Immune-Mediated Nephropathy and Systemic Autoimmunity in Mice Does Not Require Receptor Interacting Protein Kinase 3 (RIPK3). PLoS ONE, 11(9), e0163611.

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Multiparametric Flow Cytometry Analysis

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Non-viable cells were excluded from all analyses using Live/Dead eF780 dye (1:1000, eBioscience). Surface staining was performed in PBS containing 0.5% FCS and 2 mM EDTA. Intracellular cytokine staining (ICCS) was performed on cells stimulated with PMA (10 ng/ml) and ionomycin (1 µg/ml) in the presence of GolgiStop (BD Bioscience, 1:1000) for 4 hr. Cells were surface stained before fixation and permeabilization using eBioscience reagents. Staining for intracellular Notch1 and FoxP3 was performed after fixation and permeabilization using FoxP3 staining kit reagents (eBioscience). The following antibodies, all purchased from eBioscience and/or Biolegend, were used; CD4 Alexa700 (GK1.5, 1:100), CD69 FITC (H1.2F3, 1:100), Notch1-PE (mN1A, 1:100, Biolegend), CD8a APC (53–6.7, 1:200), CD19 (1D3, 1:200), B220 FITC (RA3-6B2, 1:100), Vb8.1/2 FITC (KJ16-133, 1:100), FoxP3 PE (FJK-16S, 1:100, eBioscience), CD25 PE-Cy7 (PC61.5, 1:300), IFNγ PE-Cy7 (XMG1, 1:400), IL-2 eF450 (JES6-5H4, 1:100) and IL-17A PE or PE-Cy7 (17B7, 1:2–400).
Soluble IL-17A was detected in culture supernatant by ELISA using Ready-Set-Go ELISA kit (eBioscience).

Britton G.J., Ambler R., Clark D.J., Hill E.V., Tunbridge H.M., McNally K.E., Burton B.R., Butterweck P., Sabatos-Peyton C., Hampton-O’Neil L.A., Verkade P., Wülfing C, & Wraith D.C. (2017). PKCθ links proximal T cell and Notch signaling through localized regulation of the actin cytoskeleton. eLife, 6, e20003.

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Lymphocyte Immunophenotyping by Flow Cytometry

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Lymphocyte stainings were performed by incubation for 20 min on ice using conjugated the following mAbs (eBioscience, San Diego, CA, USA): CD8-FITC (#11-0081-82), CD4-FITC(#MCD0401), CXCR3-APC (#17-1831-82), CCR5-PE (#12-1951-81), CD62L-PE (#12-0621-81), CD44-APC (#17-0441-81), CD25-APC (#17-0251-82), B220 FITC (#11-0452-82), CD5-PE (#12-0051-82), CD23-APC (#MCD2305).

Baur J., Otto C., Steger U., Klein-Hessling S., Muhammad K., Pusch T., Murti K., Wismer R., Germer C.T., Klein I., Müller N., Serfling E, & Avots A. (2018). The Transcription Factor NFATc1 Supports the Rejection of Heterotopic Heart Allografts. Frontiers in Immunology, 9, 1338.

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Fluorescent Antibody Flow Cytometry

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Fluorescently labeled mAbs for flow cytometry were used as follows: FITC-B220, eFluor 450-F4/80, APC eFluor 780-CD8, Alexa Fluor 700-CD4, APC-Foxp3, and PerCP-Cy5.5-CD44 (eBioScience), and Foxp3/Transcription Factor Fixation/Permeabilization (eBioscience, 501129060). Unlabeled TCR Cβ-specific H57-597 was expressed and purified from the B-cell hybridoma (a gift from Kappler/Marrack lab). All peptides were synthesized by Peptide 2.0 Inc.

Li W., Li R., Wang Y., Zhang Y., Tomar M.S, & Dai S. (2022). Calcitonin gene-related peptide is a potential autoantigen for CD4 T cells in type 1 diabetes. Frontiers in Immunology, 13, 951281.

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Multiparameter Flow Cytometry Analysis

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After tetramers staining, the cells were stained with surface fluorescentrly labeled mAbs as follows: FITC-B220, eFluor 450-F4/80, APC eFluor 780-CD8, Alexa Fluor 700-CD4⁺, and PerCP-Cy5.5-CD44 (eBioScience). After MHC II- peptide tetramer staining and cell surface staining, cells were fixed with 4% paraformaldehyde (Thermo Scientific Cat: 00-5523, CA, US.). Intracellular staining with Foxp3-APC was done using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Cat: 00-5523, CA, US.) according to the manufacturer’s instructions. Stained single-cell suspensions were analyzed using a Fortessa flow cytometer running FACSDiva (BD Biosciences, US). Flow Cytometry Data File Standard Version 3.0 (FSC 3.0) files were analyzed with Flowjo 10.0 Software.

Li W., Zhang Y., Li R., Wang Y., Chen L, & Dai S. (2022). A Novel Tolerogenic Antibody Targeting Disulfide-Modified Autoantigen Effectively Prevents Type 1 Diabetes in NOD Mice. Frontiers in Immunology, 13, 877022.

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