Siglecf pe

Manufactured by BioLegend

SiglecF-PE is a fluorescently labeled antibody that recognizes the Siglec-F protein, a cell surface receptor expressed on eosinophils. It can be used for the identification and analysis of eosinophils in flow cytometry applications.

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5 protocols using siglecf pe

Quantifying IgE and Immune Cells in BALF

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The blood samples were collected by cardiac puncture, and centrifuged at 3000 rpm for 5 min at 4 °C. IgE level was measured by an ELISA kit (BD Bioscience) according to the manufacturer’s instructions.
After the left lung was removed, 1 mL of iced PBS was slowly injected into the trachea with a syringe. The bronchoalveolar lavage fluid (BALF) was washed several times, counted on a blood cell counting plate, and 5×10⁵ cells were added to the 7AAD-percP (BD Biosciences), CD45-eflour 450 (Biolegend), CD11b-ITC (Biolegend), and SiglecF-PE (Biolegend), according to the specification. Antibody surface staining of BALF cells. After 30 min of light staining on ice, 200 µL PBS was added, the solution was centrifugated at 5000 r/min for 5 min, and excess antibody was washed off. The cells were resuspended in 300 μL of PBS or a 1:1 mixture of PBS and 4% paraformaldehyde and then transferred to a flow tube for on-machine detection using flow cytometry (ACEA Novo Cyte^TM).

Qin W., Wang T., Liu G., Sun L., Han W, & Gao Y. (2021). Dynamic Urinary Proteome Changes in Ovalbumin-Induced Asthma Mouse Model Using Data-Independent Acquisition Proteomics. Journal of Asthma and Allergy, 14, 1355-1366.

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Multiparameter Flow Cytometry Analysis

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Thoracic cavity, spleen, BAL and skin cells were analyzed by flow cytometry. Cells were isolated as described above and subsequently blocked with PBS/1% BSA including 0.1% rat IgG (Sigma) and stained. Flow cytometric analysis was performed using a combination of the following surface markers: CD4-FITC, CD8-APC, SiglecF-PE, F4/80-PerCP-Cy5.5, CD11b-APC-Cy7 (all BioLegend), and Ly6G-PE-Cy7 (ThermoFisher). Thoracic cavity and spleen CD4⁺ T cells and CD8⁺ T cells were identified as CD4^high or CD8^high cells, respectively; neutrophils as Ly6G^high, CD4^low; eosinophils as SiglecF^high, F4/80^low; macrophage populations were identified as F4/80^high, SiglecF^low. BAL macrophages were identified as F4/80^high, SiglecF^high and neutrophils as Ly6G^high/CD11b^high. Cells were further analyzed with MHCII-PE (BioLegend) in a different panel. Cells were stained and after another wash and centrifugation step cells were analysed by flow cytometry. Analysis was performed using a BD FACS Canto system and data was subsequently analyzed using the FACS Diva 5.1 software (BD Biosciences). During analysis, cut-offs were set using the fluorescence minus one (FMO) approach. The gating strategy is shown in S1 Fig.

Frohberger S.J., Fercoq F., Neumann A.L., Surendar J., Stamminger W., Ehrens A., Karunakaran I., Remion E., Vogl T., Hoerauf A., Martin C, & Hübner M.P. (2020). S100A8/S100A9 deficiency increases neutrophil activation and protective immune responses against invading infective L3 larvae of the filarial nematode Litomosoides sigmodontis. PLoS Neglected Tropical Diseases, 14(2), e0008119.

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Lung Immune Cell Profiling by Flow Cytometry

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Lungs were aseptically removed and placed in ice-cold PBS before processing for flow cytometry. Lungs were then minced and digested using Liberase TM (Sigma-Aldrich) as described previously (54 (link)). ACK lysis buffer was used to lyse the red blood cells, and then total number of viable cells enumerated using trypan blue exclusion. Cell counts were obtained using a TC20 automated cell counter (Bio-Rad). Single-cell suspensions were then stained with Zombie Near-IR (BioLegend) to determine live and dead cells and with the following antibodies for characterization of innate immune populations: CD45 BUV385, CD11b BV510, CD11c–PerCP-Cy5.5, CX3CR1 BV785, SiglecF-PE, CD103 BV711, Ly6C-APC, Ly6G AF700 (BioLegend). Data acquisition was conducted on a Symphony Flow Cytometer (BD Biosciences) and data analyzed using FlowJo 10 (BD Biosciences). The gating scheme used for determining lung cell populations is provided in Supplemental Figure 3.

Jessop F., Schwarz B., Scott D., Roberts L.M., Bohrnsen E., Hoidal J.R, & Bosio C.M. (2022). Impairing RAGE signaling promotes survival and limits disease pathogenesis following SARS-CoV-2 infection in mice. JCI Insight, 7(2), e155896.

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Multiparameter Flow Cytometry Identification

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Cells were stained in a staining buffer containing 2% heat-inactivated rat sera, 1 μg anti-mouse CD16/32 (clone: 93, BioLegend), and fluorochrome-conjugated antibodies in the dark for 20 minutes at 4°C. Eosinophils were identified using CD45 Alexa Fluor^® 700 (clone: 30-F11, BioLegend), CD11b Brilliant violet (BV) 421™ (clone: M1/70, BioLegend), Ly6C FITC (clone: HK1.4, BioLegend), Ly6G APC Cy7 (clone: 1A8, BioLegend), and Siglec-F PE (clone: S17007L, BioLegend). ILC2s were characterised using CD45 Alexa Fluor^® 700 (clone: 30-F11, BioLegend), lineage cocktail PE (CD3ϵ clone: 145-2C11, Ly-6G/Ly-6C clone: RB6-8C5, CD11b clone: M1/70, CD45R/B220 clone: RA3-6B2, TER-119 clone: Ter-119, BioLegend), IL-7Rα (CD127) PE Cy7 (clone: A7R34, BioLegend), ICOS APC (clone: C398.4A, BioLegend), ST2 (IL-33Rα) BV421™ (clone: DIH9, BioLegend), and KLRG1 BV510™ (clone: 2F1/KLRG1, BioLegend). 7-aminoactinomycin D (7-AAD) staining was used to identify non-necrotic (‘Live’) cells. Cells were acquired on an LSRFortessa (BD Biosciences) and FACS Aria I (BD Biosciences) for cell sorting. Single-stained and unstained controls were used to compensate for spectral overlap. Data were analyzed using FlowJo^© V10 (Treestar, Ashland, OR).

Chetty A., Darby M.G., Pillaye J., Taliep A., Cunningham A.F., O’Shea M.K., Katawa G., Layland L.E., Ritter M, & Horsnell W.G. (2023). Induction of Siglec-FhiCD101hi eosinophils in the lungs following murine hookworm Nippostrongylus brasiliensis infection. Frontiers in Immunology, 14, 1170807.

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Flow Cytometric Cell Phenotyping

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Cell phenotyping was performed on an LSRII flow cytometer using FACSDiva software (BD Biosciences) and FlowJo software (Tree Star Inc.) with the following anti-mouse monoclonal antibodies: CD45-PE-Cy7, CD3-APC, CD4-A700, CD8-PB, NK1.1-PE, CD11b-APC-Cy7, B220-FITC, SiglecF-PE, F4/80-A700, CD11c-FITC, CD11b-PE-TR, Gr1-PB, and MHCII-APC-Cy7 (Biolegend). Fluorescence-activated cell sorter-based cell sorting was performed on a FACSAria III cell sorter (BD Biosciences).

Engel O., Akyüz L., da Costa Goncalves A.C., Winek K., Dames C., Thielke M., Herold S., Böttcher C., Priller J., Volk H.D., Dirnagl U., Meisel C, & Meisel A. (2015). Cholinergic Pathway Suppresses Pulmonary Innate Immunity Facilitating Pneumonia After Stroke. Stroke, 46(11).

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