Cd117 2b8

Manufactured by BioLegend

CD117 (2B8) is a mouse monoclonal antibody that recognizes the CD117 antigen, also known as c-Kit receptor. CD117 is a type III receptor tyrosine kinase that plays a critical role in the development and function of various cell types, including hematopoietic stem cells, mast cells, and melanocytes.

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Lab products found in correlation

Anti ige r35 72, by BD (3 mentions) Lsrfortessa machine, by BD (3 mentions) Cd11b m1 70, by BioLegend (2 mentions) δ tcr ebiogl3, by Thermo Fisher Scientific (2 mentions) Streptavidin bv605, by BioLegend (2 mentions) Conjugation kit, by Thermo Fisher Scientific (1 mentions) Epcam g8, by BioLegend (1 mentions) Lsrii fortessa, by BD (1 mentions) Cd11c n418, by BioLegend (1 mentions) Cd103 2e7, by BioLegend (1 mentions) Facsaria fusion cell sorter, by BD (1 mentions) Cd4 gk1, by BioLegend (1 mentions) Thy1.2 53 2 1, by BioLegend (1 mentions) Cd31 390, by Thermo Fisher Scientific (1 mentions) Mmcp 1 rf6.1, by Thermo Fisher Scientific (1 mentions) Canto 2, by BD (1 mentions) Cd8 53 6, by BioLegend (1 mentions) Cd45 30 f11, by BioLegend (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Fixation and permeabilization kit, by Thermo Fisher Scientific (1 mentions)

5 protocols using cd117 2b8

Multicolor Flow Cytometric Analysis

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For detection and analysis, the following antibodies/clones were used: FcɛR1a (MAR-1; BioLegend); CD117 (2B8; BioLegend); CD45 (30-F11; BioLegend), β7 integrin (M293; BD Biosciences); CD11b (M1/70; BioLegend); CD11c (N418; BioLegend); CD4 (GK1.5; BioLegend); CD8 (53–6.7; BioLegend); CD103 (2E7; BioLegend); EPCAM (G8.8; BioLegend); Thy1.2 (53–2.1; BioLegend); CD31 (390; eBioscience); TGF-β1 (Tw7-16B4; BioLegend); and mMCP-1 (RF6.1; eBioscience). Anti-mMCP-1 and isotype control were conjugated in parallel to Alexa Fluor 647 using a conjugation kit (Life Technologies). Intracellular staining was conducted using a BD Cytofix/Cytoperm kit (BD Bioscience), according to the manufacturer-supplied protocol. For flow cytometry, the cells were collected and stained for surface markers for 45 min before fixation. Intracellular mMCP-1 staining was conducted overnight at 4°C. All cell sorting was on a BD FACSAria Fusion cell sorter using BD FACSDiva software. For all flow cytometry not involving cell sorting, data were collected on a BD LSRII Fortessa or BD CANTO-II using BD FACSDiva software. All downstream data analysis was conducted in FlowJo.

Derakhshan T., Samuchiwal S.K., Hallen N., Bankova L.G., Boyce J.A., Barrett N.A., Austen K.F, & Dwyer D.F. (2020). Lineage-specific regulation of inducible and constitutive mast cells in allergic airway inflammation. The Journal of Experimental Medicine, 218(1), e20200321.

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Cell Isolation and Identification from Skin

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Cell isolation from the back skin was performed as previously described.³³ (link) Cells were preincubated with FcγR-specific blocking mAb (2.4G2) and washed before staining with the following mAbs: B220 (RA3-6B2), CD3 (17A2), CD4 (GK1.5), CD11c (N418), CD19 (1D3), CD45 (30F11), CD90.2 (53-2.1), Gr1 (RB6-8C5), and δ TCR (ebioGL3) from eBioscience (San Diego, Calif); CD11b (M1/70), F4/80 (BM8), and CD117 (2B8) from Biolegend (San Diego, Calif); and anti-IgE (R35-72) from BD Biosciences (San Jose, Calif). BV605 streptavidin from Biolegend was used to detect biotinylated antibodies. Cells were analyzed by flow cytometry by using an LSRFortessa machine (BD Biosciences). The data were analyzed with FlowJo software. CD4⁺ T cells (CD45⁺CD3⁺CD4⁺), γδ TCR⁺ T cells (CD45⁺CD3⁺δTCR⁺), eosinophils (CD45⁺CD3^–GR1⁺SiglecF⁺), basophils (CD45⁺CD3^–α–IgE⁺CD117^–), MCs (CD45⁺CD3^–α–IgE⁺CD117⁺), ILCs (CD45⁺CD3^–Lin^–CD90⁺), and ILC2s (CD45⁺CD3^–Lin^– CD90⁺GATA3⁺) in the skin were identified as shown in Fig E1 (in the Online Repository available at www.jacionline.org).

Leyva-Castillo J.M., Galand C., Mashiko S., Bissonnette R., McGurk A., Ziegler S.F., Dong C., McKenzie A.N., Sarfati M, & Geha R.S. (2020). ILC2 activation by keratinocyte-derived IL-25 drives IL-13 production at sites of allergic skin inflammation. The Journal of Allergy and Clinical Immunology, 145(6), 1606-1614.e4.

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Flow Cytometric Characterization of Skin Cells

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Cell isolation from the back skin was performed as previously described^{33 (link)}. Cells were preincubated with FcγR-specific blocking mAb (2.4G2) and washed before staining with the following monoclonal antibodies (mAbs):B220 (RA3–6B2), CD3 (17A2), CD4 (GK1.5), CD11c (N418), CD19 (1D3), CD45 (30F11), CD90.2 (53–2.1), Gr1 (RB6–8C5) and δ TCR (ebioGL3) from eBioscience, CD11b (M1/70), F4/80 (BM8) and CD117 (2B8) from Biolegend and anti-IgE (R35–72) from BD Biosciences. BV605 Streptavidin from Biolegend was used to detect biotinylated antibodies. Cells were analyzed by flow cytometry using an LSRFortessa machine (BD Biosciences). The data was analyzed with FlowJo software. CD4⁺ T cells (CD45⁺CD3⁺CD4⁺), γδ TCR⁺ T cells (CD45⁺CD3⁺δTCR⁺), eosinophils (CD45⁺CD3⁻GR1⁺SiglecF⁺), basophils (CD45⁺CD3α−IgE⁺CD117⁻), mast cells (CD45⁺CD3⁻α−IgE⁺CD117⁺), ILCs (CD45⁺CD3⁻Lin⁻CD90⁺) and ILC2s (CD45⁺CD3⁻Lin⁻ CD90⁺GATA3⁺) in the skin were identified as shown in Fig E1.

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Surface Marker Analysis of Immune Cells

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For surface markers analysis, live cells were re-suspended in 1 × PBS and stained with anti-mouse CD45 (30-F11, Biolegend), CD11b (M1/70, R&D Systems), F4/80 (BM8, Biolegend), CD86 (GL-1, Biolegend), MHC-II (M5/114.15.2, Biolegend), CD117 (2B8, Biolegend), CD115 (AFS98, Biolegend), CD16/32 (93, Biolegend), CD34 (HM34, Biolegend), Sca1 (D7, Biolegend), SIRPα (P84, biolegend), Lin (Stem Cell), and APC-Cy7 Streptavidin (Biolegend) at 4°C for 30 min. The concentration at each antibody was used as the product protocol recommended. For intracellular cytokine staining, cells were fixed and permeabilized with Fixation and Permeabilization Kit (eBioscience) at room temperature for 40 min and labeled with anti-mouse CD206 (C068C2, Biolegend). Multicolor FACS analysis was performed on an LSR Fortessa Analyzer (BD Biosciences). All data analysis was performed using the flow cytometry analysis program FlowJo-V10.

Zhao C.C., Han Q.J., Ying H.Y., Gu X.X., Yang N., Li L.Y, & Zhang Q.Z. (2022). TNFSF15 facilitates differentiation and polarization of macrophages toward M1 phenotype to inhibit tumor growth. Oncoimmunology, 11(1), 2032918.

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Characterizing Skin Cell Immune Phenotypes

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Skin cells were preincubated with FcγR-specific blocking mAb (2.4G2) and washed before staining with the following monoclonal antibodies (mAbs): CD3 (17A2), CD4 (GK1.5), CD45 (30F11) and δ TCR (ebioGL3) from eBioscience, CD11b (M1/70), and CD117 (2B8) from Biolegend and anti-IgE (R35–72) from BD Biosciences. For cytokine staining, cells were stimulated with Ionomycin (0.5ug/ml; Sigma), Phorbol 12,13-dibutyrate (1ug/ml; Sigma), Brefeldin A (eBioscience), Monensin (eBioscience) in complete RPMI for 3 hours before surface staining. Then, cells were fixed and permeabilized (BD Biosciences Cytofix/Cytoperm) and stained in permeabilization solution with IL-22 (1H8PWSR), from Ebiosciences. Cells were analyzed by flow cytometry using an LSRFortessa machine (BD Biosciences). The data was analyzed with FlowJo software.

Leyva-Castillo J.M., Sun L., Wu S.Y., Rockowitz S., Sliz P, & Geha R. (2022). Single cell transcriptome profile of mouse skin undergoing antigen driven allergic inflammation recapitulates findings in atopic dermatitis skin lesions. The Journal of allergy and clinical immunology, 150(2), 373-384.

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