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Anti siglecf

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Anti-SiglecF is a laboratory reagent used for the detection and analysis of Siglec-F, a cell surface receptor expressed on certain immune cells. It is a specific antibody that binds to Siglec-F, allowing for the identification and quantification of cells expressing this receptor.

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Lab products found in correlation

Anti cd11b, by BioLegend (5 mentions) Anti cd11c, by BD (5 mentions) Anti cd45, by BioLegend (5 mentions) Anti cd45, by BD (5 mentions) Anti cd4, by BD (4 mentions) Anti ly6g, by BD (4 mentions) Anti ccr3, by BD (3 mentions) Facscanto, by BD (3 mentions) Anti cd11c, by BioLegend (3 mentions) Anti cd8a, by BioLegend (3 mentions) Anti cd4, by BioLegend (3 mentions) Facscalibur, by BD (3 mentions) Anti cd11b, by BD (3 mentions) Anti cd45, by Thermo Fisher Scientific (2 mentions) Kaluza, by Beckman Coulter (2 mentions) Anti gr 1, by BD (2 mentions) Cellquest, by BD (2 mentions) Phycoerythrin conjugated anti cd107a, by Thermo Fisher Scientific (2 mentions) Anti fcεri, by Thermo Fisher Scientific (2 mentions) Facsaria 2, by BD (2 mentions)

21 protocols using anti siglecf

1

Phospho-ERK1/2 Activation in Eosinophils

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Total bone marrow cells were stained with anti-CD45 (ebioscience), anti–Siglec-F (BD Bioscience) and anti-CCR3 (BD Bioscience). The mature eosinophils (triple positive) were sort by MoFlo XDP (Beckman Coulter). The cells were activated with bronchoalvelolar lavage fluid (BALF) which was obtained from the lungs of CC10-Il13Tg/Pirb+/+ mice for the indicated time points (0, 5 and 10 minutes), and cells were fixed in 4% paraformaldehyde/PBS and permeabilized using saponin-based permeabilization buffer (X1, invitrogen). Cells were stained with phospho-ERK1/2 (Cell signaling). Events were acquired by FACSCanto (BD Bioscience), and data were analyzed using the Kaluza (BeckmanCoulter) or FlowJo (TreeStar) software. For phosphoflow analysis, the mean fluorescence intensity (MFI) for each time point, in each biological repeat, was normalized to baseline and expressed as the fold change over baseline.
Ben Baruch-Morgenstern N., Mingler M.K., Stucke E., Besse J.A., Wen T., Reichman H., Munitz A, & Rothenberg M.E. (2016). Paired immunoglobulin-like receptor B inhibits IL-13–driven eosinophil accumulation and activation in the esophagus. Journal of immunology (Baltimore, Md. : 1950), 197(3), 707-714.
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2

Multiparameter Flow Cytometry of Immune Cells

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Flow cytometric analysis of bone marrow, peripheral blood cells or enzymatically digested esophagus was conducted using the following antibodies: anti-CD11b (R&D), anti–GR-1 (BD Bioscience), anti–Siglec-F (BD Bioscience), anti-CCR3 (BD Bioscience), anti–PIR-A/B (ebioscience), IgG2b (ebioscience), anti-CD45 (ebioscience) and anti-CD11c (BD Bioscience). Cell counts were conducted using 123count beads (ebioscience) according to the manufacturers’ instructions. In all experiments, at least 50,000 events were acquired by (FACSCalibur, BD Bioscience), and data were analyzed using the Kaluza (BeckmanCoulter) or FlowJo (TreeStar) softwares.
Ben Baruch-Morgenstern N., Mingler M.K., Stucke E., Besse J.A., Wen T., Reichman H., Munitz A, & Rothenberg M.E. (2016). Paired immunoglobulin-like receptor B inhibits IL-13–driven eosinophil accumulation and activation in the esophagus. Journal of immunology (Baltimore, Md. : 1950), 197(3), 707-714.
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3

Isolation and Transfer of Eosinophils

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Eosinophils were isolated from the peritoneal cavity of 4get/IL-5tg mice using the Mouse Streptavidin Rapidspheres Isolation Kit and EasySep magnet (Stemcell Technologies), according to the manufacturer's instructions. The following biotinylated antibodies were applied for negative selection: anti CD45R, anti-Ter-119, anti-CD4, anti-CD8, anti-Ly6C and anti-MHC II (all from ebiosience). The purity of eosinophils was determined by flow cytometry as IL-4/eGFP+ SiglecF+ (PE-labelled anti-SiglecF; 1:400; BD Pharmingen) cells with an average of 90–95%. Isolated eosinophils (2 × 107) or vehicle control was injected intra-orbital into ΔdblGATA mice 5 days after K/BxN serum transfer. Eosinophil transfer was confirmed by flow cytometry gating on IL-4/eGFP+ SiglecF+ cells in the blood 7 days post serum transfer.
Chen Z., Andreev D., Oeser K., Krljanac B., Hueber A., Kleyer A., Voehringer D., Schett G, & Bozec A. (2016). Th2 and eosinophil responses suppress inflammatory arthritis. Nature Communications, 7, 11596.
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4

Comprehensive Immune Cell Profiling

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Cells were incubated with Fc-block (clone 2.4G2, BioXcell) and stained with the following antibodies (clones) from BioLegend, eBioscience or BD Biosciences: anti-CD3 (clone 145–2C11), anti-CD4 (RM4.5), anti-CD8 (53–6.7), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD45 (30-F11), anti-Ly6C (HK1.4), anti-Ly6G (1A8), anti-SiglecF (1RNM44 N), and anti-CCR3 (J073E5). Fixable viability dyes, such as Ghost Dyes (Tonbo) and ViaKrome 808 (Beckman Coulter) were used to exclude dead cells from the analyses. For intracellular cytokine staining, cells were pulsed for 4 h with PMA (phorbol 12-miristate 13-acetate, 50 ng/mL), ionomycin (500 ng/mL), and Brefeldin A (GolgiPlug, BD), followed by 4% paraformaldehyde fixation and permeabilized (BD Biosciences) before intracellular cytokine staining for GM-CSF (MP1–22E9), IFN-γ (XMG1.2) and IL-17A (TC11–18H10.1). Stained cells were collected on MACSQuant analyzer (Miltenyi Biotec, Germany) or CytoFLEX (Beckman Coulter, Brea, CA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
Bing S.J., Silver P.B., Jittayasothorn Y., Mattapallil M.J., Chan C.C., Horai R, & Caspi R.R. (2020). Autoimmunity to neuroretina in the concurrent absence of IFN-γ and IL-17A is mediated by a GM-CSF-driven eosinophilic inflammation. Journal of autoimmunity, 114, 102507.
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5

Quantification of Degranulation and Eosinophils in Nasal Mucosa

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Single cell suspensions from nasal mucosa were resuspended in PBS/0.1% bovine serum albumin (BSA) seeded in a 96-well plate at 106 cells/ml, blocked with 5% goat serum and stained with Phycoerythrin conjugated anti-CD107a (eBioscience, San Diego, California, USA) and anti-Siglec-F (BD biosciences) for the detection of total degranulation40 (link) and eosinophils, respectively. For in vitro assessment of BMMC expression of FCεRI, cells were stained with anti-FCεRI (eBioscience, San Diego, California, USA) and cell viability was determined by propidium iodide staining (Sigma Aldrich, Rehovot, Israel). Data were analyzed by a FACSCalibur and CellQuest software (Becton Dickinson, San Jose California, USA).
Karra L., Haworth O., Priluck R., Levy B.D, & Levi-Schaffer F. (2014). Lipoxin B4 promotes the resolution of allergic inflammation in the upper and lower airways of mice. Mucosal immunology, 8(4), 852-862.
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6

Identification of Human and Mouse ILC2

Cited 2 times
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Human peripheral blood ILC2 were identified as CD45+Lin−IL-7Rα+CRHT2+ cells. Mouse lung ILC2 were identified as CD45+Lin−Thy1.2+ST2+ cells according to our previous studies.7 (link)-9 (link) Lineage antibodies for human cells included anti-CD16 (3G8), anti-CD11c (3.9), anti-CD123 (6H6), anti-CD19 (HIB19), anti-CD3 (UCHT1), anti-CD5 (L17F12), anti-CD94 (DX22), anti-CD34 (581). Additional antibodies used to recognize human ILC2 included anti-CRTH2 (BM16), anti-CD127 (A019D5) and anti-CD117 (104D2). Lineage antibodies for mouse ILC2 included anti-B220 (RA3-6B2), anti-CD3 (2C11), anti-TCRβ (H57), anti-TCRγδ (GL-3), anti-CD11b (M1/70), anti-CD5 (53-7.3). Additional anti-mouse antibodies used included anti-IL-5 (TRFK5), anti-IL-13 (eBio13A), anti-Thy1.2 (53-2.1), anti-Ki67 (16A8), anti-Gr-1 (RB6-8C5), anti-CD11c (N418), anti-CD25 (PC61.5), anti-ST2 (DJ8; MD Bioproducts) and anti-Siglec F (E50-2440; BD Biosciences). Antibodies were purchased from Biolegend or eBioscience, unless specified otherwise. FACS was performed on a FACSAria II (BD Biosciences) and regular flow cytometric analysis was performed on a FACSCanto (BD Biosciences). The Intracellular Cytokine Staining Kit from BD Pharmingen was used to stain intracellular IL-13 and IL-5 as we previously described. 7 (link)-9 (link)
Ye L., Pan J., Liang M., Pasha M.A., Shen X., D’Souza S.S., Fung I.T., Wang Y., Patel G., Tang D.D, & Yang Q. (2020). A critical role for c-Myc in group 2 innate lymphoid cell activation. Allergy, 75(4), 841-852.
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7

Single Cell Immunophenotyping of Lung and Brain

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Lung and brain single cell suspensions were quantified by the trypan blue exclusion method. Cells were blocked with anti‐CD16/32 (Biolegend, San Diego, CA); immune cell types were identified using combinations of antibody listed in Table 1. All antibodies were purchased from Biolegend, except anti‐Siglec F, which was purchased from BD Pharmingen (Franklin Lakes, NJ). LIVE/DEAD Fixable Dead Cell Stain was used to exclude dead cells (Thermo Fisher Scientific). Samples were run on a LSRFortessa (BD Biosciences) using FACSDiva software version 8.0, and analyzed using FlowJo version 9.9.6.
Farris B.Y., Monaghan K.L., Zheng W., Amend C.D., Hu H., Ammer A.G., Coad J.E., Ren X, & Wan E.C. (2019). Ischemic stroke alters immune cell niche and chemokine profile in mice independent of spontaneous bacterial infection. Immunity, Inflammation and Disease, 7(4), 326-341.
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8

Colonic Leukocyte Phenotyping by Flow Cytometry

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Colonic LP cells were incubated with anti-mouse CD16/CD32 (BioLegend) to block non-specific Fc receptors, followed by a cell surface staining with the corresponding mixture of fluorescently-labelled monoclonal antibodies. Seven-amino actinomycin D (BioLegend) was used to discriminate live and dead cells. The following antibodies conjugated with biotin, PE, peridinin chlorophyll-Cy5.5, PE-Cy7, APC, APC-Cy7, brilliant violet (BV) 421, BV510, BV650, BV711, or BV785 were used for flow cytometry: anti-B220, anti-CD4, anti-IA/IE, anti-Ly6G, anti-Siglec F (all from BD Biosciences), anti-CD3e, anti-CD8a, anti-CD11b, anti-CD11c, anti-CD45, anti-CD45.2, anti-CXCR4, anti-F4/80, anti-TER119 (all from BioLegend), anti-Ly6C, anti-NK-1.1, anti-Siglec F (all from Miltenyi Biotec, Auburn, CA) and anti-CCR2 (R&D Systems). Following extracellular staining, the cells were fixed and permeabilised with Cytofix/Cytoperm (BD Biosciences) and sequentially incubated with rabbit anti-collagen type I (Rockland, Limerick, PA), followed by Alexa Fluor 647-conjugated goat anti-rabbit IgG (Invitrogen). Data were acquired on LSRFortessa and processed using FlowJo software (Tree Star, Ashland, OR) with appropriate isotype controls to determine gating. Cell sorting was performed using FACSAria II (BD Biosciences).
Kuroda N., Masuya M., Tawara I., Tsuboi J., Yoneda M., Nishikawa K., Kageyama Y., Hachiya K., Ohishi K., Miwa H., Yamada R., Hamada Y., Tanaka K., Kato T., Takei Y, & Katayama N. (2019). Infiltrating CCR2+ monocytes and their progenies, fibrocytes, contribute to colon fibrosis by inhibiting collagen degradation through the production of TIMP-1. Scientific Reports, 9, 8568.
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9

Multi-antibody Panel for Cell Sorting

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Monoclonal, murine-specific antibodies from BioLegend included: anti-CD11c (N418), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5), anti-CD19 (6D5), anti-CD4 (RM4–5), anti-CD8α (53-6.7), anti-CD25 (PC61), anti-NK1.1 (PK136), anti-Thy1.2 (53-2.1), anti-CD45.2 (104), anti-EpCAM (G8.8), and anti-CD45 (30-F11). Antibodies from eBioscience included: anti-CD11b (M1/70), anti-CD4 (RM4–5), anti-CD8α (53-6.7), anti-Thy1.2 (53-2.1), anti-F4/80 (BM8), anti-CD49b (DX5), anti-CD5 (53-7.3), anti-CD45 (30-F11), anti-IRF4 (3E4), anti-GATA3 (TWAJ), anti-B220 (RA3-6B2), and anti-CD31 (390). Antibodies from BD Biosciences included: anti-CD11c (HL3), anti-CD25 (PC61), anti-Thy1.2 (53-2.1), anti-Ly6G (1A8), anti-α4β7 (DATK32), and anti-Siglec-F (E50-2440). An Alexa Fluor 488-conjugated anti-Siglec-F antibody was generated using purified anti-Siglec-F with an Alexa Fluor 488 monoclonal antibody labeling kit (Life Technologies).
Exclusion of DAPI (4',6-diamidine-2'-phenylindole dihydrochloride; Roche) or LiveDead (Life Technologies) identified live cells, which were enumerated with flow cytometric CountBright Absolute beads (Life Technologies), according to the manufacturer’s instructions. Sample data were acquired with a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc.). Cells were sorted using a MoFlo XDP (Beckman Coulter) instrument.
Mohapatra A., Van Dyken S.J., Schneider C., Nussbaum J.C., Liang H.E, & Locksley R.M. (2015). Group 2 innate lymphoid cells utilize the IRF4-IL-9 module to coordinate epithelial cell maintenance of lung homeostasis. Mucosal Immunology, 9(1), 275-286.
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10

Eosinophil Identification Protocol

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Cells (3×105) were incubated first with mouse FcBlock (2.4G2; BD PharMingen, San Diego, CA, USA) to inhibit nonspecific binding of antibodies. After washing, cells were stained with anti-Siglec F, anti-Gr-1, anti-CD11b, anti-CD11c and respective isotype controls (BD Biosciences, PharMingen). Numbers of positive cells were quantified by flow cytometry (FACSCanto flow cytometer, BD Biosciences, San Jose, CA). Eosinophils were identified as SiglecF+Gr-1+CD11b+CD11c-[21] (link), [38] (link). Data were collected on a FACSCanto flow cytometer and analysed with FlowJo software (version 10, Tree Star, Inc).
Yang M., Eyers F., Xiang Y., Guo M., Young I.G., Rosenberg H.F, & Foster P.S. (2014). Expression Profiling of Differentiating Eosinophils in Bone Marrow Cultures Predicts Functional Links between MicroRNAs and Their Target mRNAs. PLoS ONE, 9(5), e97537.
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