Fcεri

Manufactured by BioLegend

Sourced in United States

FcεRI is a high-affinity receptor for the Fc region of IgE antibodies. It plays a central role in the activation of mast cells and basophils, initiating the release of inflammatory mediators in response to allergen exposure.

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PE anti-FcεRI FITC‐anti‐FcεRI FcεRI-FITC FcεRI (Mar-1, CD-117/c-kit-FcεRI+, FITC-conjugated anti-FcεRI FITC-conjugated anti-mouse FcεRI α FITC-conjugated anti-FcεRI (MAR-1, Biotinylated anti-FcεRI (clone MAR-1, Biotin-conjugated anti-FcεRI antibodies

9 protocols using fcεri

Assessing PDI Expression in Activated Mast Cells

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BMMCs and MC/9 cells (a murine mast cell line) were incubated with mAbs against mouse c-Kit (Biolegend), FcεRI (Biolegend), PDI (Life Technologies), and isotype controls for PDI (IgG2a/Life Technologies). Expression of cell surface PDI was assessed at various times in cells after activation with IgE and antigen and in unactivated controls. Intracellular PDI expression was assessed 30 min later in fixed and permeabilized cells. Flow cytometric analysis was performed using an Accuri C6 flow cytometer and Flow jo software.

Krajewski D., Polukort S.H., Gelzinis J., Rovatti J., Kaczenski E., Galinski C., Pantos M., Shah N.N., Schneider S.S., Kennedy D.R, & Mathias C.B. (2020). Protein Disulfide Isomerases Regulate IgE-Mediated Mast Cell Responses and Their Inhibition Confers Protective Effects During Food Allergy. Frontiers in Immunology, 11, 606837.

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Flow Cytometric Immune Cell Profiling

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Staining for flow cytometry was carried out as described previously (7 (link), 15 (link)). Fluorophore-labeled antibodies were: human lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56), FcεRI, CRTH2, IL7Rα, CD25, IL5 from Biolegend; PFVD eFluor 780, pSTAT3, pSTAT5 from e-Bioscience; ST2 and IL25 receptor (IL17RB) from R&D Systems; ID3 and MEK from BD Bioscience; c-Fos from Cell signaling Technology, and glucocorticoid receptor antibody from Bioss Antibodies. The labeled FcεRI antibody was added to the lineage cocktail. We stained cell surface markers on live cells first. Then, we fixed cells using 4% paraformaldehyde, permeabilized them with 0.1% saponin and performed staining for intracellular molecules (cytokines, signaling molecules and transcription factors). Flow cytometry was performed on LSRII (BD Biosciences). The data was analyzed by the FlowJo software. Isotype antibody controls and FMO (fluorescence minus one) were used to develop the gating and data analysis strategies.

Liu S., Verma M., Michalec L., Liu W., Sripada A., Rollins D., Good J., Ito Y., Chu H., Gorska M.M., Martin R.J, & Alam R. (2017). Steroid Resistance of Airway Type 2 Innate Lymphoid Cells (ILC2s) from Severe Asthma: The Role of Thymic Stromal cell Lymphopoietin (TSLP). The Journal of allergy and clinical immunology, 141(1), 257-268.e6.

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Comprehensive Mouse Spleen and Bone Marrow Immunophenotyping

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Briefly, mouse spleens and BM were collected and dissociated through a 40μm strainer to produce a single cell suspension. Red blood cells were lysed using 0.86% ammonium chloride (Sigma). Cells were counted, washed in flow cytometry buffer (1% BSA (Sigma), 2mM EDTA (Invitrogen) in PBS. FcR were blocked with 2.4G2 (BioXCell) and Rat IgG (Invitrogen) while staining with McAb at 4⁰ for 30 minutes. Intracellular staining was carried out using the eBioscience kit. Samples were acquired using a FACSCanto II or LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Inc.). The following mAb against mouse antigens were used as FITC, PE, PerCP-Cy5.5, PE-Cy7, allophycocyanin (APC), APC-ef780, Pacific blue, AF450, AF700, PE Texas Red, or biotin conjugates: FcεRI, AA4.1, CD23, CD43, IgM, FcγRII/III, ckit, Sca-1, CD34, CD138, CD45R (B220; RA3-6B2), CD278 (ICOS; C398.4A), IgD (11–26) (eBioscience), CD4 (RM4–5), CD8a (53–6.7), CD95 (Fas; Jo2), CXCR5 (2G8) (BD Biosciences), CD3 (17A2), CD19 (6D5), Foxp3, CTLA-4, Siglec F, CD11b, F4/80, TCRβ, Gr-1, FcεRI, and CD279 (PD-1; J43) (Biolegend). Biotinylated antibodies were detected using PerCP-Cy5.5– (BD Biosciences) or APC-conjugated streptavidin (eBioscience). FITC or biotin-conjugated PNA was obtained from Vector Laboratories. Plots shown are on a Logicle scale.

Zaretsky A.G., Konradt C., Dépis F., Wing J.B., Goenka R., Atria D.G., Silver J.S., Cho S., Wolf A.I., Quinn W.J., Engiles J.B., Brown D.C., Beiting D., Erikson J., Allman D., Cancro M.P., Sakaguchi S., Lu L., Benoist C.O, & Hunter C.A. (2017). T Regulatory Cells Support Plasma Cell Populations in the Bone Marrow. Cell reports, 18(8), 1906-1916.

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Multiparametric Flow Cytometry Analysis

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Cells stained with live/dead fixable blue dead cell staining kit (ThermoFisher Scientific) were blocked with anti-CD16/32 antibody (eBioscience) and stained with specified antibodies. For intracellular staining, cells were fixed and permeabilized with fixation/permeabilization working solution (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Antibodies used included those recognizing CD107a, c-kit, phospho-Syk, ST2, CD127 (eBioscience), FcεRI (Biolegend), and thymic stromal lymphopoietin receptor (TSLPR; R&D Systems, Minneapolis, MN, USA). Cells were acquired on a BD LSR Fortessa cytometer (BD Biosciences). Data was analyzed using the Flow-Jo software (TreeStar Inc., Ashland, OR, USA).

Benedé S., Cody E., Agashe C, & Berin M.C. (2018). Immune Characterization of Bone Marrow-Derived Models of Mucosal and Connective Tissue Mast Cells. Allergy, Asthma & Immunology Research, 10(3), 268-277.

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Murine Bone Marrow Mast Cell Differentiation

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Bone marrow cells were collected from femurs from 4-to–8-month-old mice and cultured in Dulbecco's Modified Eagle Medium with glucose and L-glutamine, supplemented with 10% fetal bovine serum, penicillin/streptomycin, and sodium pyruvate (all from Gibco™, ThermoFisher Scientific, Waltham, MA, USA) plus 25 ng/mL recombinant murine stem cell factor (SCF) (all cytokines and growth factors were from Peprotech, Rocky Hill, NJ, USA) and 30 ng/mL interleukin (IL)-3. Bone marrow MCs were differentiated into either a MMCs with additional IL-9 at 5 ng/mL and transforming growth factor (TGF)-β at 1 ng/mL or a CTMC with IL-4 at 1 ng/mL. MCs were cultured in 75-cm² tissue culture flasks, incubated at 37℃ in a humidified incubator under 5% (v/v) CO₂ for a minimum of 4 weeks and up to 8 weeks before they were used for functional assays. Twice a week, the medium was changed by transferring the cell suspension to a 50-mL conical polypropylene centrifuge tube, and centrifuging for 10 minutes at 200×g, at room temperature. The culture flasks were changed every time the medium was changed. The maturity and purity of the cells were examined by flow cytometric analysis for the expression of c-Kit (eBioscience, San Diego, CA, USA) and FcεRI (Biolegend, San Diego, CA, USA).

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Enrichment and Culture of ILC3

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LP cells were obtained and pooled from RAG KO mice, and ILC3 were enriched by negative selection with a cocktail of biotinylated Abs specific for CD11b, CD11c, F4/80, Gr-1, Ter119, NK1.1, FcεRI, EpCAM, and KLRG1 (Biolegend) followed by incubation with Anti-Biotin MicroBeads and magnetic separation as directed (Miltenyi Biotec). The negative fraction was stained with Fixable Live/Dead dye (Invitrogen), CD45 and CD90 specific Abs, and fluorescently conjugated streptavidin (to detect any remaining biotin labeled cells) (Biolegend). CD45+ CD90+ streptavidin- live cells (ILC3-enriched ILCs) were FACS sorted to ~97% purity using a FACSAria II (BD Biosciences). 20,000 cells per well were cultured 20 hours at 37°C in V-bottom plates (in IMDM media described above). Recombinant mouse cytokines concentrations used were 40ng/ml IL-23, 10ng/ml IL-2, and 40ng/ml TNFα (R&D Systems).

Paustian A.M., Paez-Cortez J., Bryant S., Westmoreland S., Waegell W, & Kingsbury G. (2017). Continuous IL-23 stimulation drives ILC3 depletion in the upper GI tract and, in combination with TNFα, induces robust activation and a phenotypic switch of ILC3. PLoS ONE, 12(8), e0182841.

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Murine Bone Marrow-Derived Mast Cell Isolation

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Bone marrow cells were obtained from femurs and tibias of 6–8-week-old C57BL/6 female and male mice. Cells were maintained in RPMI-1640 supplemented with 10% FBS, 5 μM β-mercaptoethanol (Life Technologies, USA) and 2% antibiotic and antimycotic (Sigma, USA) (complete RPMI 1640 medium), plus 10 ng/mL of murine recombinant IL-3 (Peprotech, USA) and 10 ng/mL of murine recombinant stem cell factor (SCF) (Peprotech, USA). Non-adherent cells were transferred to fresh culture medium twice a week for 6 − 9 weeks. The purity of bone marrow-derived mast cells (BMMC) was ≥ 90% as assessed by flow cytometry after staining of CD117 (clone: 2B8, BioLegend, USA; 0.25 μg/mL) and FcεRI (clone: MAR R-1, BioLegend, USA; 0.16 μg/mL).

Soria-Castro R., Meneses-Preza Y.G., Rodríguez-López G.M., Ibarra-Sánchez A., González-Espinosa C., Pérez-Tapia S.M., Flores-Borja F., Estrada-Parra S., Chávez-Blanco A.D, & Chacón-Salinas R. (2022). Valproic acid restricts mast cell activation by Listeria monocytogenes. Scientific Reports, 12, 15685.

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Mast Cell Reconstruction in Sash Mice

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The MC reconstruction in Sash mice was followed the protocol (Wolters et al., 2005 (link)), briefly, bone marrow derived mast cells (BMMCs) were generated as follows: bone marrow cells from 8-week-old C57BL/6 mice were harvested and incubated in RPMI-1640 supplemented with 1% non-essential amino acids, 50 mg/L gentamycin, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% fetal bovine serum, 10ng/mL murine IL-3, 30 ng/mL murine stem cell factor and 0.1% 2-mercaptoethanol. Medium was changed every week. Cells were harvested for reconstruction after 3-week incubation. The purity of the BMMCs has a MC phenotype higher than > 90% as determined by flow cytometry immunostaining with FcεRI (BioLegend) and c-Kit (eBioscience). For reconstruction, 3-week-old Sash mouse was injected i.v. with BMMCs (5 × 10⁶) and maintained for an additional 4 weeks before usage.

Song S.T., Wu M.L., Zhang H.J., Su X, & Wang J.H. (2022). Mast Cell Activation Triggered by Retrovirus Promotes Acute Viral Infection. Frontiers in Microbiology, 13, 798660.

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Mast Cell Derivation from Mouse Bone Marrow

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Bone marrow-derived mast cells (BMMC) are commonly obtained by culturing with combinations of IL-3 and SCF. However, as reports have shown that chronic exposure to SCF results in attenuated FceRI signaling and degranulation of mast cells, here we have used cultures of bone marrow from 6-8 week-old C57BL/6 female mice in the presence of IL-3 alone, as described [11, 12] . Mast cell purity was ≥95% as determined by flow cytometry after staining of CD117 (clone: 2B8, BioLegend, USA) and FcεRI (clone: MAR-1, BioLegend, USA). The protocol to derive mast cells from mouse bone marrow was reviewed and approved by the Committee for Ethics in Research, ENCB-IPN.

Rodríguez-López G.M., Soria-Castro R., Campillo-Navarro M., Pérez-Tapia S.M., Flores-Borja F., Wong-Baeza I., Muñoz-Cruz S., López-Santiago R., Estrada-Parra S., Estrada-García I., Chávez-Blanco A.D, & Chacón-Salinas R. (2020). The histone deacetylase inhibitor valproic acid attenuates phospholipase Cγ2 and IgE-mediated mast cell activation. Journal of leukocyte biology, 108(3).

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