F4 80

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

The F4/80 is a mouse cell surface marker that is commonly used to identify and isolate macrophages. It is a glycoprotein that is expressed on the surface of mature macrophages, as well as some other myeloid cell types. The F4/80 antibody can be used for flow cytometry, immunohistochemistry, and other applications to detect and study macrophages in various experimental systems.

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Anti-F4/80 microbeads (34 citations) Anti-F4/80 MicroBeads UltraPure (17 citations) F4/80 microbeads (12 citations) Anti-F4/80 Microbeads Ultrapure Kit (6 citations) Anti-F4/80 (6 citations) F4/80 magnetic beads (4 citations) Anti-F4/80 antibody (4 citations) F4/80 MicroBeads UltraPure (3 citations) Anti-F4/80-FITC/anti-FITC-microbeads (3 citations) F4/80 antibody (2 citations)

13 protocols using f4 80

Characterizing Purity of Bone Marrow-Derived Macrophages

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The purity of the BMM population was determined via flow cytometry analysis. 1x10⁶ cells were taken from the freshly harvested BMMs, processed and stained with the required antibodies according to the manufacturer’s protocol. The fluorescently labeled monoclonal antibodies (mAbs) that specifically recognize proteins expressed by macrophages were used for phenotypical characterization. The used two-color panel included two surface antigens, F4/80 (1:100, Miltenyi Biotec, Germany) and CD11b (1:100, Miltenyi Biotec, Germany). In this two-color immunofluorescence protocol, the samples were single stained with each antibody, and then stained using both antibodies. Data were acquired with a MACS-Quant device (Miltenyi Biotec, Germany) using the MACSQUANTIFY™ software (Figure 1A).

Barcena M.L., Niehues M.H., Christiansen C., Estepa M., Haritonow N., Sadighi A.H., Müller-Werdan U., Ladilov Y, & Regitz-Zagrosek V. (2021). Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli. Frontiers in Immunology, 12, 758767.

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Characterization of 4-1BB Expression on Lung Cells

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Single-cell suspensions of lungs were prepared from mice as previously described (22 (link)). To reduce non-specific antibody binding, cells were incubated with a Fc receptor block (BD Pharmingen, San Jose, CA, USA). Then lung single-cell suspensions were stained with antibodies to CD45 (BD Pharmingen), F4/80 (Miltenyi Biotech, Bergisch Gladbach, Germany), CD11c (BD Pharmingen), CD137 (eBioscience™, San Diego, CA, USA), CD137L (eBioscience™), CD4 (BD Pharmingen), CD44 (BD Pharmingen), and CD62L (BD Pharmingen). For MH-S cells, the cell surface was stained by CD137 or CD137L antibodies after Fc blocking. Isotype-matched mAb were used as negative controls. A FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo V10 software were used to analyze the expression of 4-1BB on lung cells.

Lu Y., Li C., Du S., Chen X., Zeng X., Liu F., Chen Y, & Chen J. (2018). 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice. Frontiers in Immunology, 9, 1848.

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Antigen-Specific CD8+ DC Targeting

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C57BL/6 mice were injected i.d. with 100 µg FITC-labelled AntpMAPMUC1tetFITC or PBS in footpads for 18 h. Popliteal lymph nodes cells were isolated and pooled from mice (n = 3) and cells labelled with fluorochrome-conjugated anti-CD3, anti-CD19, anti-CD11c and anti-F4/80 (BD Pharmingen) to separate T cells, B cells, CD11c^hi DC and F4/80^hi macrophages respectively [45 (link),48 (link)].
To assess binding of DC subsets popliteal lymph nodes were pooled (n = 4) and resuspended in free balanced salt solution (BSS) with 0.1 M EDTA (pH 7.2) to dissociate T cells from DCs and incubated with CD11c microbeads (10 µL beads/10⁷ cells) (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at 4 °C. Cells were washed and resuspended in BSS and passed through AutoMACS columns (Miltenyi Biotech, Germany). Purity of cells (CD11c⁺) was >90%.
To differentiate DC subpopulations, cells were stained with biotin-labelled anti-DEC205 and streptavidinPE-Cy7 and anti-CD8-PE together with anti-CD11cAPC and analysed for FITC expression. Three major subsets in the popliteal lymph nodes subsets were identified: CD8^– DEC205^– (immature), CD8⁺ DEC205^– (immature) and CD8^low DEC205⁺ DCs (migratory) [46 (link)].

Brooks N., Hsu J., Esparon S., Pouniotis D, & Pietersz G.A. (2018). Immunogenicity of a Tripartite Cell Penetrating Peptide Containing a MUC1 Variable Number of Tandem Repeat (VNTR) and A T Helper Epitope. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(9), 2233.

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Tumor Immune Cell Phenotyping

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Tumors were disaggregated and digested in collagenase D and DNase for 30 min at 37°C to obtain a single-cell suspension. After neutralizing unspecific binding with a CD16/CD32 antibody (clone 93), single-cell suspensions were stained with fixable Live/Dead staining (Life Technologies), followed by incubation with specific monoclonal antibodies diluted 1:100 (primary antibodies directly conjugated) to assess the phenotype. The antibodies used were: CD45 (REA737, Miltenyi Biotec); Ly-6G (1A8, Biolegend); Ly6C (REA796, Miltenyi Biotec), CD11b (REA592, Miltenyi Biotec); F4/80 (REA126, Miltenyi Biotec), CD19 (REA749, Miltenyi Biotec), CD206 (C068C2, Biolegend), CD11c (REA754, Miltenyi Biotec), B220 (RA3-6B2, Biolegend), CD3 (REA641, Miltenyi Biotec), CD8 (REA601, Miltenyi Biotec), CD4 (REA604, Miltenyi Biotec), NK1.1 (REA1162, Miltenyi Biotec), NKp44 (P44-8 BioLegend), NKp46 (29A1.4, BD). For flow gating, we used isotype controls of fluorescence minus one control. All the antibodies were purchased from Miltenyi Biotec or Biolegend. Samples were acquired on a BD Canto-II flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (TreeStar, Ashland, OR). The respective RRID of each reagent is listed in the key resources table.

Colucci M., Zumerle S., Bressan S., Gianfanti F., Troiani M., Valdata A., D’Ambrosio M., Pasquini E., Varesi A., Cogo F., Mosole S., Dongilli C., Desbats M.A., Contu L., Revankdar A., Chen J., Kalathur M., Perciato M.L., Basilotta R., Endre L., Schauer S., Othman A., Guccini I., Saponaro M., Maraccani L., Bancaro N., Lai P., Liu L., Pernigoni N., Mele F., Merler S., Trotman L.C., Guarda G., Calì B., Montopoli M, & Alimonti A. (2024). Retinoic acid receptor activation reprograms senescence response and enhances anti-tumor activity of natural killer cells. Cancer Cell, 42(4), 646-661.e9.

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Macrophage Differentiation Assay

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To analyze the macrophagic differentiation of monocytes, cells were stained with CD11b (#130-110-554, Miltenyi), F4/80 (#130-116-499, Miltenyi), and CD206 (#141717, Biolegend, San Diego, CA, USA). Finally, cells were washed and fixed in 2% paraformaldehyde. Fluorescence was measured with a MACSQuant^® Analyzer.

Parigiani M.A., Ketscher A., Timme S., Bronsert P., Schlimpert M., Kammerer B., Jacquel A., Chaintreuil P, & Reinheckel T. (2020). Conditional Gene Targeting Reveals Cell Type-Specific Roles of the Lysosomal Protease Cathepsin L in Mammary Tumor Progression. Cancers, 12(8), 2004.

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Comprehensive Immune Cell Profiling of PVA Sponge

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For the analysis of cells recruited to the PVA sponge model used for the harvest of EVs, cells were subjected to flow cytometry using Fc block (Cat#130-092-575, Miltenyi Biotec, San Diego, CA, USA), followed by staining with antibodies specific for the following immune cell markers from Miltenyi Biotec. (CD11b, #130-109-287; CD11c, #130-110-840; CD45, #130-110-803; CD44, #130-119-127; F4/80, #130-102-422; Gr1, #130-102-233; Ly6G, #130-107-912, Ly6C, #130-123-796, MHCII, # 130-119-122; CD4, #130-118-696, and CD3, #130-117-788). Propidium iodide (#130-093-233, Miltenyi Biotec, San Diego, CA, USA) was used to exclude dead cells. Isotype antibodies were used for all fluorescence studies. (VioBlue/PacBlue, #130-113-454; VioGreen/BV510, #130-113-456; FITC, #130-113-449; PE, #130-113-450; PE-Vio770, #130-113-452; APC, #130-113-446; and APC-Vio770/Fire750, #130-113-447, Miltenyi Biotec, CA, USA). All flow cytometry was performed on a MACSQuant 10 instrument (Miltenyi Biotec, San Diego, CA, USA) and analyzed using FlowJo software (Version 10.7.1, Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Park D.J., Duggan E., Ho K., Dorschner R.A., Dobke M., Nolan J.P, & Eliceiri B.P. (2022). Serpin-loaded extracellular vesicles promote tissue repair in a mouse model of impaired wound healing. Journal of Nanobiotechnology, 20, 474.

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Isolation and Polarization of Mouse Bone Marrow-Derived Macrophages

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Culture of mice BMDMs was performed as previously described [67 (link)]. Briefly, both femurs and tibias of mice were collected and washed in sterile PBS. Both ends of femurs and tibias were removed, and bone marrow cavities were flushed with RPMI 1640 medium (Gibco, Big Cabin, OK, USA). Cell suspension was centrifuged at 200× g for 6 min at room temperature (RT). Afterward, cells were collected and cultured in RPMI 1640 medium containing 10% FBS, 20% supernatants of L929 mouse fibroblasts as conditioned medium providing macrophage colony-stimulating factor (M-CSF). The medium was replenished every 2–3 days. Cells were collected as BMDMs on day 6, stained with antibodies of CD11b (Miltenyi, 130113806, Bergisch Gladbach, Germany) and F4/80 (Miltenyi, 130117509, Bergisch Gladbach, Germany), and analyzed through flow cytometry (BD, Franklin Lakes, NJ, USA).
For induction of polarization, BMDMs (1 × 10⁶) were stimulated with either 1 μg/mL LPS or 20 ng/mL IL-4 for 48 h in the presence or absence of MSCs-CM (from 2 × 10⁵ MSCs). BMDMs were then collected, stained with antibodies of CD206 (Biolegend, cat no: 141712, San Diego, CA, USA) and CD80 (Miltenyi, cat no: 130102883, Bergisch Gladbach, Germany), and analyzed through flow cytometry.

Xu Z., Lin L., Fan Y., Huselstein C., De Isla N., He X., Chen Y, & Li Y. (2022). Secretome of Mesenchymal Stem Cells from Consecutive Hypoxic Cultures Promotes Resolution of Lung Inflammation by Reprogramming Anti-Inflammatory Macrophages. International Journal of Molecular Sciences, 23(8), 4333.

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Murine Peritoneal Macrophage Isolation

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Mice were injected i.p. with 10 µg/kg LPS and euthanized 4 hours later by cardiac puncture to collect blood while anesthetized with 2.5% isofluorane, followed by cervical dislocation. The mouse peritoneum was exposed and injected with 3 mL PBS containing 0.5% BSA and 2mM EDTA. The peritoneal lavage fluid was collected and centrifuged for 10 min at 400 x g to pellet cells. Cells were then stained with fluorochrome conjugated REAfinity antibodies (2 µL per 10⁶ cells) against mouse CD11b (VioGreen), F4/80 (PE), and Ly6G (APC) (Miltenyi Biotec) for 15 minutes at 4°C. Cells were washed and centrifuged before FACS analysis on a BD LSR II. Analysis of the FACS data was performed with FlowJo software.

Reeves A.R., Sansbury B.E., Pan M., Han X., Spite M, & Greenberg A.S. (2021). Myeloid-specific deficiency of long-chain acyl CoA synthetase 4 (Acsl4) reduces inflammation in vitro and in vivo by remodeling phospholipids and reducing production of arachidonic acid-derived proinflammatory lipid mediators. Journal of immunology (Baltimore, Md. : 1950), 207(11), 2744-2753.

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Multicolor Flow Cytometry Profiling of Myeloid Cells

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Single-cell suspensions derived from mouse tumors or human Mφs were stained with fluorescence dye-conjugated antibodies against CD11b (1:100; BioLegend 101206 or 101211), CD206 (1:100; BioLegend, 321110, 141707, or BD Biosciences, 551135), IL-10 (1:50; BD, 566568), F4/80 (1:100; Miltenyi Biotec, 130-102-327), IFN-γ (1:50; BioLegend, 502505), IL-10 (1:50; BioLegend, 505009), arginase 1 (1:50; eBioscience, 17-3697-80), or control immunoglobulin G (IgG). Cells were analyzed by using Accuri C6 (BD Biosciences) and FACSCanto II flow cytometers (BD Biosciences). The data were analyzed by FlowJo software (V10).

Yang F., Akhtar M.N., Zhang D., El-Mayta R., Shin J., Dorsey J.F., Zhang L., Xu X., Guo W., Bagley S.J., Fuchs S.Y., Koumenis C., Lathia J.D., Mitchell M.J., Gong Y, & Fan Y. (2024). An immunosuppressive vascular niche drives macrophage polarization and immunotherapy resistance in glioblastoma. Science Advances, 10(9), eadj4678.

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Histological and Flow Cytometric Analysis of Tumor Inflammation

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Formalin-fixed paraffin-embedded tissues were used for histology and immunohistochemistry. Two pathologists, blinded to experimental group, evaluated sections for pathology and inflammation. Inflammation was scored on a 0–4 scale (0, normal mucosa; 1, minimal inflammation (occasional scattered granulocytes and leukocytes); 2, mild inflammation (scattered granulocytes with occasional infiltrates); 3, moderate inflammation (scattered granulocytes with patchy infiltrates); and 4, severe inflammation (multiple extensive areas with abundant granulocytes and marked infiltrates), as previously described (9 (link)).
Immunohistochemistry was performed in EDTA antigen retrieval buffer, as previously described (6 ), using antibodies against CD11b (Abcam), F4/80 (Abcam), and Ly6G (Novus Biologicals). DAB-positive cells in tumor-positive fields of view were acquired at 10X magnification and quantified using Viziopharm acquisition and analysis software (Hoersholm, Denmark), and expressed as the number of positive cells in each histologically defined area. Single cell suspensions were generated from spleen and cervical lymph nodes from tumor-bearing mice for flow cytometry. Cells were stained according to manufacturer’s protocol against CD11b-APC, Ly6C-FITC, Ly6G-Pacific Blue, and F4/80-PE with at least 10,000 events analyzed by Miltenyi MACSQuant cytometer and software.

Zhang X., Hyer J.M., Yu H., D’ Silva N.J, & Kirkwood K.L. (2014). DUSP1 phosphatase regulates the proinflammatory milieu in head and neck squamous cell carcinoma. Cancer research, 74(24), 7191-7197.

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