Anti siglecf e50 2440

Manufactured by BD

Sourced in United States

Anti-SiglecF (E50-2440) is a laboratory reagent used in flow cytometry applications. It is an antibody that specifically binds to the mouse SiglecF protein, which is expressed on the surface of certain immune cells. The product can be used to identify and quantify cells expressing SiglecF, but its precise applications and intended uses are not specified.

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Lab products found in correlation

Anti cd11b m1 70, by BioLegend (5 mentions) Anti cd11c n418, by BioLegend (5 mentions) Anti cd3 17a2, by BioLegend (4 mentions) Anti cd45 30 f11, by BioLegend (4 mentions) Anti cd11c hl3, by BD (3 mentions) Anti ly6g 1a8, by BD (3 mentions) Anti cd19 6d5, by BioLegend (3 mentions) Anti nk1.1 pk136, by BioLegend (3 mentions) Anti cd45.2 104, by BioLegend (3 mentions) Anti ly6g 1a8, by BioLegend (3 mentions) Anti tcrβ h57 597, by BioLegend (3 mentions) Anti il 13 ebio13a, by Thermo Fisher Scientific (2 mentions) Anti cd8 53 6.7, by BioLegend (2 mentions) Anti cd45.1 a20, by BioLegend (2 mentions) Anti cd44 im7, by BioLegend (2 mentions) Anti ki67 sola15, by Thermo Fisher Scientific (2 mentions) Aurora spectral cytometer, by Tree Star (2 mentions) Anti human vβ5.1 lc4, by Thermo Fisher Scientific (2 mentions) Anti vβ11 kt11, by BioLegend (2 mentions) Anti mouse cd185 cxcr5 l138d7, by BioLegend (2 mentions)

16 protocols using anti siglecf e50 2440

Multicolor Flow Cytometry Analysis

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All antibodies (Abs) used were purchased from BioLegend unless specified otherwise. Abs in the lineage (Lin) cocktail included anti-FcεR (MAR-1; eBioscience), anti-B220 (RA3-6B2), anti-CD19 (ID3; BD Biosciences), anti-Mac-1 (M1/70; BD Biosciences), anti-Gr-1 (R86-8C5; BD Biosciences), anti-CD11c (HL3; BD Biosciences), anti-NK1.1 (PK 136), anti-Ter-119 (Ter-119), anti-CD3 (145-2C11), anti-CD8α (53-6.7; BD Biosciences), anti-CD5 (53-7.3), anti-TCRβ (H57-597), and anti-γδTCR (GL-3; BD Biosciences). Additional Abs used included anti-CD45.2 (104), anti-CD45.1 (A20), anti-CD4 (RM4-5; BD Biosciences), anti-cKit (2B8), anti-Sca-1(D7), anti-Thy1.2 (53-2.1), anti-IL-5 (TRFK5), anti-IL-13 (eBio13A; eBioscience), anti-Siglec F (E50-2440; BD Biosciences), anti-IL7Rα (A019D5), anti-α4β7 (DATK32; eBioscience), anti-CD25 (PC61), anti-PLZF (Mags.21F7; eBioscience), and anti-ST2 (DIH9).
Cell sorting was performed on a FACSAria II (BD Biosciences), and flow cytometric analysis was performed on a LSR-II (BD Biosciences). Intracellular staining of transcription factors was done using Foxp3 Staining Buffer kit (eBioscience). Staining of cytokines was carried out using Cytofix/cytoperm kit (BD Bioscience).

Wang H.C., Qian L., Zhao Y., Mengarreli J., Adrianto I., Montgomery C.G., Urban JF J.r., Fung K.M, & Sun X.H. (2017). Down-regulation of E protein activity augments an ILC2 differentiation program in the thymus. Journal of immunology (Baltimore, Md. : 1950), 198(8), 3149-3156.

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Multiparameter Flow Cytometry of Mouse Blood

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Flow cytometry was performed on mouse blood as previously described (63 (link)). Briefly, to obtain single-cell suspensions, spleen samples were ground between sterile frosted glass slides in 7 ml of red blood cell lysis buffer (0.15 mM NH₄Cl, 10 mM KHCO3, 0.1 mM disodium ethylenediaminetetraacetic acid, pH 7.2) and then filtered through sterile nylon mesh. Whole blood, obtained by cardiac puncture, was treated with red blood cell lysis buffer twice. Cell pellets were washed and resuspended in phosphate-buffered saline containing 2 mM ethylenediaminetetraacetic acid and 0.5% bovine serum albumin. Fluorophore-conjugated antibodies with specificity to mouse cell antigens were as follows: anti-CD11b (M1/70) (BD Biosciences Cat# 550993, RRID:AB_394002), anti-CD11c (HL3) (BD Biosciences Cat# 561022, RRID:AB_2033997), anti-SiglecF (E50-2440) (BD Biosciences Cat# 552125, RRID:AB_394340), anti-Ly6G (1A8) (BD Biosciences Cat# 561236, RRID:AB_10611860), and anti-CD36 (JC63.1) (Cayman Chemical Cat# 10009870, RRID:AB_10342682). Live/dead fixable dead cell stains (ThermoFisher) were used to exclude dead cells. CD11b+/c+ cells were analyzed for CD36 expression. Paraformaldehyde-fixed cells were acquired using a Becton Dickinson (BD) LSR Fortessa flow cytometer (BD Biosciences, University of Alberta Flow Cytometry core) and analyzed with FlowJo (version 10) software (RRID:SCR_008520).

Rekhi U.R., Omar M., Alexiou M., Delyea C., Immaraj L., Elahi S, & Febbraio M. (2021). Endothelial Cell CD36 Reduces Atherosclerosis and Controls Systemic Metabolism. Frontiers in Cardiovascular Medicine, 8, 768481.

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Single-Cell Tumor Tissue Analysis

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To obtain single-cell suspensions, tumor tissue samples were digested with collagenase IV (1 mg/ml; Sigma-Aldrich) and deoxyribonuclease (DNase) I (0.2 mg/ml; Sigma-Aldrich). The following fluorochrome-conjugated antibodies were used: anti-CCR3 (J073E5), anti-CD45 (30-F11), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD103 (2E7), anti-CD64 (X54-5/7.1), anti-CD3 (17A2), anti-CD8 (53-6.7), anti-CD4 (GK1.5), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD69 (H1.2F3), anti-CD90.1 (OX-7), anti-CD90.2 (30-H12), anti-F4/80 (BM8), anti–Gr-1 (RB6-8C5), anti-GZMB (GB11), anti–Ly-6C (HK1.4), anti-MHCII (M5/114.15.2), anti-NK1.1 (PK136), and anti-TCR Vβ13 (MIR12-4) (all from BioLegend); anti–Siglec-F (E50-2440) (BD Biosciences); and anti-IFNγ (XMG1.2), anti-perforin (eBioOMAK-D), and Fixable Viability Dye eFluor 780 (65-0865) (from eBioscience). Data were collected using a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software.

Cheng J.N., Luo W., Sun C., Jin Z., Zeng X., Alexander P.B., Gong Z., Xia X., Ding X., Xu S., Zou P., Wan Y.Y., Jia Q., Li Q.J, & Zhu B. (2021). Radiation-induced eosinophils improve cytotoxic T lymphocyte recruitment and response to immunotherapy. Science Advances, 7(5), eabc7609.

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Comprehensive Immune Cell Profiling

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For cell sorting and flow cytometry analysis, cell suspension were prepared as described above, incubated with fixable viability dye and subsequently stained with following fluorophore-conjugated monoclonal antibodies: anti-CD45 (30-F11, BD Biosciences), anti-CD45.2 (104, Biolegend), anti-CD90.2 (thy1.2) (53–2.1, Biolegend), anti-CD3 (17A2, Biolegend), anti-CD4 (Biolegend, RM4–5), anti-CD8 (53–6.7, Biolegend), anti-CD11b (M1/70; Biolegend), anti-CD11c (N418, Biolegend), anti-Ly6C (HK1.4, Biolegend), anti-Ly6G (1A8, BD Biosciences), anti-Siglec-F (E50–2440, BD Bioscience), anti-mouse MHC Class II (I-A/I-E) (M5/114.15.2, eBioscience), anti-NK1.1 (PK136, Biolegend), anti-CD19 (6D5, Biolegend), anti-T1/ST2 (DJ8, MD Biosciences), anti-KLRG1 (2F1, Biolegend), anti-FoxP3 (FJK-16S, eBiosciences), anti-Ki-67 (16A8, Biolegend), anti-Gata3 (TWAJ, eBioscience), anti-IL13 (eBio13A, eBioscience), anti-IFNgR1 (XMG1.2, BD Bioscience).

Cautivo K.M., Matatia P.R., Lizama C.O., Mroz N.M., Dahlgren M.W., Yu X., Sbierski-Kind J., Taruselli M.T., Brooks J.F., Wade-Vallance A., Caryotakis S.E., Chang A.A., Liang H.E., Zikherman J., Locksley R.M, & Molofsky A.B. (2022). Interferon gamma constrains type 2 lymphocyte niche boundaries during mixed inflammation. Immunity, 55(2), 254-271.e7.

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Multiparametric Flow Cytometry Profiling

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For cell surface staining, cells were pre-incubated with 2.4G2 Fcγ RII/RIII blocking mAb for 15 minutes and then stained with the appropriate combinations of mAbs diluted in HBSS + 2% FBS for 30 minutes at 4°C. Cells were analyzed on a BD FACS CantoII, or Cytek Aurora Spectral Cytometer and data were processed with FlowJo software (TreeStar). The complete list of antibodies use is as follows: anti-human Vβ5.1 (LC4, Invitrogen), anti-TCRβ (H57–597, BioLegend), anti-CD44 (IM7, BioLegend), anti-Vβ11 (KT11, BioLegend), anti-CD45.2 (104, BioLegend), anti-CD45.1 (A20, BioLegend), anti-SiglecF (E50–2440, BD Bioscience), anti-CD45 (30-F11, BioLegend), anti-CD11c (N418, BioLegend), anti-CD11b (M1/70, BioLegend), anti-Ly6G (1A8, BioLegend), anti-CD19 (6D5, BioLegend), anti-NK1.1 (PK136, BioLegend), anti-CD3 (17A2, BioLegend), Zombie Red (BioLegend), anti-mouse CD185/CXCR5 (L138D7, Biolegend), anti-mouse CD279/PD-1 (29F.1A12, Biolegend), and anti-Ki67 (SolA15, Invitrogen).

Morgun E., Zhu J., Almunif S., Bobbala S., Aguilar M.S., Wang J., Conner K., Cui Y., Cao L., Seshadri C., Scott E.A, & Wang C.R. (2023). Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells. bioRxiv.

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Isolation and Analysis of Skin Immune Cells

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1cm² skin pieces from unmanipulated or tape stripped mice, were obtained. Skin pieces were finely chopped using scissors after fat removal and digested for 90 minutes in the media containing Liberase (0.2mg/mlRoche) and DNAse II (Sigma), with continuous shaking at 37° C. Digested skin homogenates were filtered, washed and resuspended in PBS and used for flow cytometry. Cells were preincubated with FcγR-specific blocking mAb (2.4G2) and washed before staining with the following monoclonal antibodies (mAbs): CD3 (17A2), CD45 (30F11), Gr1 (RB6-8C5) from eBioscience, CD11b (M1/70) from Biolegend and anti-Siglec-F (E50-2440) from BD Biosciences. Cells were analyzed by flow cytometry using an LSRFortessa machine (BD Biosciences).

Leyva-Castillo J.M., McGurk A, & Geha R. (2020). Allergic skin inflammation and S. aureus skin colonization are mutually reinforcing.. Clinical immunology (Orlando, Fla.), 218, 108511.

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Characterization of Immune Cell Surface Phenotype

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To characterize the cell surface phenotype, isolated cells were resuspended in PBS containing 10% FCS, 20 mM HEPES, and 10 mM EDTA. After blocking Fc receptors with anti-mouse CD16/CD32 (2.4G2, BD Biosciences, San Diego, CA, USA) for 15 min at 4 °C, the cells were stained for 30 min at 4 °C with the following antibodies: mAb against CCR3 (83101) from R&D Systems (Minneapolis, MN, USA); anti-SiglecF (E50-2440) from BD Biosciences; anti-CD45 (30-F11), F4/80 (BM8), CD3 (145-2C11), and B220 (RA3-6B2) from BioLegend (San Diego, CA, USA); and anti-CD4 (RM4-5) and CD8 (53–6.7) from eBioscience (San Diego, CA, USA). Each sample was analysed with a FACSCalibur™ flow cytometer (BD Biosciences) and the data were processed using FlowJo software (Tree Star, Ashland, OR, USA).

Lee E.H., Itan M., Jang J., Gu H.J., Rozenberg P., Mingler M.K., Wen T., Yoon J., Park S.Y., Roh J.Y., Choi C.S., Park W.J., Munitz A, & Jung Y. (2018). Eosinophils support adipocyte maturation and promote glucose tolerance in obesity. Scientific Reports, 8, 9894.

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Comprehensive Immune Cell Profiling

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Anti-Ly6G (1A8), anti-Ly6C (HK1.4), anti-CD45.2 (104), anti-NK1.1 (PK136), anti-CD19 (6D5), anti-Thy1.2 (53–2.1), anti-CD11b (M1/70), anti-F4/80 (BM8), anti-CD64 (X54-5/7.1), and anti-CD16/32 (93) were purchased from BioLegend. Anti-CD11c (HL3) and anti-SiglecF (E50-2440) were purchased from BD Biosciences (CA, USA). Anti-CD115 (ASF98) and anti-MHC class II (M5/114.15.2) were purchased from Thermo Fisher Scientific. Anti-CD204 (REA148) and REA control (REA293) were purchased from Miltenyi Biotech (Bergisch Gladbach, Germany). Dead cells were excluded by DAPI (Dojindo, Kumamoto, Japan).

Uchida Y., Nishitai G., Kikuchi K., Shibuya T., Asano K, & Tanaka M. (2020). CD204-positive monocytes and macrophages ameliorate septic shock by suppressing proinflammatory cytokine production in mice. Biochemistry and Biophysics Reports, 23, 100791.

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Murine Lung Cell Isolation and Characterization

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Murine lungs were chopped and incubated in RPMI1640 media with 1 mg/mL type IV collagenase (Worthington BioChem, Lakewood, NJ, USA) for at least 1 h 30 min at 37 °C. Red blood cells (RBC) were lysed using RBC lysis buffer (Biolegend, San Diego, CA, USA). Lung single cells were stained with following fluorochrome-conjugated antibodies. For surface staining, the following antibodies were used. Anti-CD45 (30-F11), Anti-CD3e (145-2C11), anti-CD11c (HL3), anti-CD11b (M1/70), anti-CD19 (ID3), anti-CD49b (DX5), anti-FcεRIα (MAR-1), anti-CD90,2 (30-H12), anti-F4/80 (BM8) and anti-Ly6G (1A8), purchased from Biolegend. Anti-SiglecF (E50-2440) was purchased from BD Bioscience (San Diego, CA, USA). For intracellular staining, the following antibodies were used: anti-IL5 (TRFK5) and anti-IL17A (TC11-18H10.1), purchased from Biolegend (San Diego, CA, USA). Anti-IL13 (eBio13A) was purchased from Thermo Fisher Scientific. For intracellular staining, a Fixation/Permeabilization Solution Kit with BD GolgiPlug (BD Biosciences, San Diego, CA, USA) was used following the manufacturer’s protocol. Flow cytometry was carried out using LSRFortessa™ X-20 (BD biosciences, San Jose, CA, USA) and analyzed by FlowJo (V10.2) software (BD biosciences, San Jose, CA, USA). The gating strategy for immune cell population was represented in Supplementary Figure S1.

Yoon S., Song S., Shin J.W., Kang S., Kim H.Y, & You H.J. (2020). Protective Effects of Korean Herbal Remedy against Airway Inflammation in an Allergic Asthma by Suppressing Eosinophil Recruitment and Infiltration in Lung. Antioxidants, 10(1), 6.

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Isolation of Alveolar Macrophages from Mice

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Mice were either infected or given PBS and euthanized 24 h post-treatment. Lungs were harvested, and dissociated using VDI 12 tissue homogeniser (VWR) in sterile PBS. Single-cell suspensions were obtained by flushing the samples through 70 μm strainer. To exclude dead cells, samples were stained with FVD eFluor 506 (eBioscience), prior to Fc blocking with anti-CD16/CD32 (2.4G2, BD). To sort out CD45⁺CD11c^highSiglecF⁺ alveolar macrophages, cell suspensions were stained for cell surface proteins using monoclonal antibodies conjugated to allophycocyanin-eFluor 780, allophycocyanin and brilliant violet 421: anti-CD45 (30-F11), anti-CD11c (N418) and anti-SiglecF (E50-2440), purchased from BD Bioscience, eBioscience and Biolegend. RNA from sorted alveolar macrophages was extracted using Power SYBR Green Cells-to-CT Kit (4402954, Ambion) according to manufacturer’s instructions.

Ivin M., Dumigan A., de Vasconcelos F.N., Ebner F., Borroni M., Kavirayani A., Przybyszewska K.N., Ingram R.J., Lienenklaus S., Kalinke U., Stoiber D., Bengoechea J.A, & Kovarik P. (2017). Natural killer cell-intrinsic type I IFN signaling controls Klebsiella pneumoniae growth during lung infection. PLoS Pathogens, 13(11), e1006696.

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