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Anti mouse f4 80

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Anti-mouse F4/80 is a laboratory reagent used for the identification and isolation of mouse macrophages. It is a monoclonal antibody that specifically binds to the F4/80 antigen expressed on the surface of mouse macrophages.

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Lab products found in correlation

Anti mouse cd45, by BD (2 mentions) Tsa fluorescein, by PerkinElmer (2 mentions) Simple stain mouse max po, by Nichirei Biosciences (2 mentions) Anti mouse ly 6g ly 6c gr 1, by BioLegend (2 mentions) Lsm 800 airyscan confocal microscope, by Zeiss (2 mentions) Blocking one, by Nacalai Tesque (2 mentions) Anti mouse ly6g ly6c, by BD (1 mentions) Anti mouse cd3e, by BD (1 mentions) Cellquest pro software, by BD (1 mentions) Facscalibur, by BD (1 mentions) Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions) Tumor dissociation kit, by Miltenyi Biotec (1 mentions) Anti mouse cd11b, by BD (1 mentions) Ly 6g gr 1, by BD (1 mentions) Cd66b, by BD (1 mentions) Molecular probe, by Thermo Fisher Scientific (1 mentions) Anti cd8, by BD (1 mentions) Anti tcrβ, by BD (1 mentions) Anti cd25, by BioLegend (1 mentions) Anti gr1, by BioLegend (1 mentions)

8 protocols using anti mouse f4 80

1

Murine Tumor Immune Profiling

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For murine samples, mice with tumors were killed according to the institutional ethical guidelines and femurs, tibias, and tumors were collected. Signal cell of suspension of tumor was obtained by using the Tumor Dissociation Kit (Miltenyi Biotech, Bergish Gladbach, Germany, 130-096-730) according to the manufacturer’s instructions. Antibodies (BD Pharmingen, San Diego, CA, USA) used are listed: anti-mouse-CD3e (BD Pharmingen, 566494), anti-mouse-CD45 (BD Pharmingen, 553079), anti-mouse-CD8e (BD Pharmingen, 553035), anti-mouse-CD11b (BD Pharmingen, 550993), anti-mouse-Ly-6G/Ly-6C (BD Pharmingen, 553129), Anti-mouse-F4/80 (BD Pharmingen, 565410). For cell apoptosis assays, apoptosis cells were measured by a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. Data were acquired using FACS Calibur (BD Biosciences), and analyzed using Cell Quest Pro software (BD Biosciences)46 (link).
Xia S., Wu J., Zhou W., Zhang M., Zhao K., Liu J., Tian D, & Liao J. (2021). SLC7A2 deficiency promotes hepatocellular carcinoma progression by enhancing recruitment of myeloid-derived suppressors cells. Cell Death & Disease, 12(6), 570.
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2

Immunostaining Reagents and Protocols

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In vitro reagents were obtained from Life Technologies (Carlsbad, CA), unless otherwise noted. Antibodies such as Alexa Fluor–conjugated anti-human CD45, CD14, CD33, and CD66b (described below) as well as anti-mouse F4/80 and Ly6g/Gr1 were purchased from BD Biosciences Inc. (San Jose, CA, USA), eBioscience (San Diego, CA, USA), BioLegend (San Diego, CA, USA), or Miltenyi Biotec (San Diego, CA, USA), as denoted below. For human/primate immunostaining, anti-human CD68 Alexa Fluor–conjugated (488 or 647) Ab was purchased from Santa Cruz Biotechnology (Dallas, TX). The same CD11b (anti-mouse/human) Ab (BioLegend) was used for both human and mouse staining. Cy3-conjugated anti-mouse αSMactin Ab was purchased from Sigma-Aldrich (St. Louis, MO). Medium PS spheres (mean, 526.6 ± 53.3 μm) (catalog no. 136, lot no. 30055) were purchased from Phosphorex (Hopkinton, MA). Material aliquots used in this study were submitted for endotoxin testing by a commercial vendor (Charles River Laboratories, Wilmington, MA), with results showing that spheres contained endotoxin levels of <0.05 EU/ml (below detectable limits) (table S1).
Doloff J.C., Ma M., Sadraei A., Tam H.H., Farah S., Hollister-Lock J., Vegas A.J., Veiseh O., Quiroz V.M., Rakoski A., Aresta-DaSilva S., Bader A.R., Griffin M., Weir G.C., Brehm M.A., Shultz L.D., Langer R., Greiner D.L, & Anderson D.G. (2023). Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response. Science Advances, 9(24), eade9488.
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3

Immunofluorescence Analysis of Skin Lesions

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Extracted skin lesions were fixed in 4% paraformaldehyde for 24 h at 4°C; the tissues were then immersed overnight in 10% sucrose, 30% sucrose, or OCT compound, followed by embedding in OCT compound. Seven-micrometer-thick sections were prepared and stained with hematoxylin and eosin (H&E), in accordance with the appropriate standard protocol. For immunofluorescence staining, the tissue slices were permeabilized and blocked in 25 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.2% saponin, 10% blocking One (Nacalai Tesque). The mycobacteria were stained with anti-Mycobacteria LAM (ViroStat, 5179) after treatment with fluorochrome-conjugated secondary antibody. To visualize neutrophils and macrophages, the following antibodies were obtained from commercial sources; anti-mouse Ly-6G/Ly-6C (Gr-1) (BioLegend, 108403) and anti-mouse F4/80 (BD, 565409), respectively. The samples were incubated with an amplifying secondary antibody (Simple stain mouse MAX-PO, Nichirei, 414311) after reacting with the primary antibody, then stained using a tyramide-based technique (TSA Fluorescein, PerkinElmer). Images were obtained on the LSM 800 Airyscan confocal microscope (Carl Zeiss). The cell numbers in two randomly selected tissue areas (magnification, x20) were calculated using the ImageJ software.
Suzuki T., Boonyaleka K., Okano T., Iida T., Yoshida M., Fukano H., Hoshino Y., Iwakura Y., Ablordey A.S, & Ashida H. (2023). Inflammasome-triggered IL-18 controls skin inflammation in the progression of Buruli ulcer. PLOS Pathogens, 19(11), e1011747.
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4

Detailed Phenotypic Analysis of Immune Cells

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The antibodies used for phenotypic analysis of the cells were purchased from ebiosciences: anti-PD-1 (clone: J43), anti- CD25 (clone: PC61.5), anti- CD4 (GK1.5), anti- MHCII (IA/IE), anti-H-2Kb and anti- PDL1 (clone: MIH5) or Biolegend: anti- Gr-1 (clone: RB6-8C5), CD11b (clone: M1/70), anti- TCRβ (clone: H57-597), anti- CD8, anti- CD4, ant- Tim-3 (clone: B8.2C12), and anti- mouse F4/80 (clone: BM8); and anti-Ly6C (clone: AL-21), anti- Ly6-G (clone: 1A8), anti-CD124 (IL-4Rα), or anti- arginase-1 from BD biosciences. The fluorescent conjugates used were as follows: phycoerithrin (PE), PE– CY7, FITC, allophycocyannin (APC), APC-CY7, pacific blue, peridinin-chlorophyll-cy5.5 (PerCP Cy5.5), and aqua amine (Molecular Probe, Invitrogen) for exclusion of dead cells. NKT cells were stained using CD1d tetramer (tet) obtained from the NIH Tetramer facility, Rockville, MD.
Hongo D., Tang X., Baker J., Engleman E.G, & Strober S. (2014). Requirement for Interactions of Natural Killer T Cells and Myeloid Derived Suppressor Cells for Transplantation Tolerance. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 14(11), 2467-2477.
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5

Immunofluorescence Analysis of Skin Lesions

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Extracted skin lesions were fixed in 4% paraformaldehyde for 24 h at 4°C; the tissues were then immersed overnight in 10% sucrose, 30% sucrose, or OCT compound, followed by embedding in OCT compound. Seven-micrometer-thick sections were prepared and stained with hematoxylin and eosin (H&E), in accordance with the appropriate standard protocol. For immunofluorescence staining, the tissue slices were permeabilized and blocked in 25 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.2% saponin, 10% blocking One (Nacalai Tesque). The mycobacteria were stained with anti-Mycobacteria LAM (ViroStat, 5179) after treatment with fluorochrome-conjugated secondary antibody. To visualize neutrophils and macrophages, the following antibodies were obtained from commercial sources; anti-mouse Ly-6G/Ly-6C (Gr-1) (BioLegend, 108403) and anti-mouse F4/80 (BD, 565409), respectively. The samples were incubated with an amplifying secondary antibody (Simple stain mouse MAX-PO, Nichirei, 414311) after reacting with the primary antibody, then stained using a tyramide-based technique (TSA Fluorescein, PerkinElmer). Images were obtained on the LSM 800 Airyscan confocal microscope (Carl Zeiss). The cell numbers in two randomly selected tissue areas (magnification, x20) were calculated using the ImageJ software.
Suzuki T., Boonyaleka K., Okano T., Iida T., Yoshida M., Fukano H., Hoshino Y., Iwakura Y., Ablordey A.S, & Ashida H. (2023). Inflammasome-triggered IL-18 controls skin inflammation in the progression of Buruli ulcer. PLOS Pathogens, 19(11), e1011747.
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6

Immune Cell Profiling in Small Intestine

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After blocking Fc receptors with anti-mouse CD16/CD32 (BD Biosciences), small intestinal epithelial cells were stained with anti-mouse Ly-6G (BioLegend), anti-mouse F4/80 (BD Biosciences), anti-mouse CD86 (BD Biosciences), anti-mouse CD206 (BD Biosciences), and anti-mouse CD45 (BD Biosciences). The stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using FlowJo Software (FlowJo, Ashland, OR). Neutrophils were defined as Ly6G+ cells and macrophages as F4/80+ cells and M1 macrophages as F4/80+ CD86+ and M2 macrophages as F4/80+ CD206+. Lymphocytes were defined as CD45+ cells.
Ho J., Chan H., Liang Y., Liu X., Zhang L., Li Q., Zhang Y., Zeng J., Ugwu F.N., Ho I.H., Hu W., Yau J.C., Wong S.H., Wong W.T., Ling L., Cho C.H., Gallo R.L., Gin T., Tse G., Yu J., Chan M.T., Leung C.C, & Wu W.K. (2020). Cathelicidin preserves intestinal barrier function in polymicrobial sepsis. Critical Care, 24, 47.
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7

Cell Death, Cycle, and VEGF Analysis

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For the cell death assay, treated melanoma tissues or macrophages were stained with R-Phycoerythrin conjugated Annexin V and 7-AAD according to the manufacturer’s protocol (BD Biosciences). Cell death was quantified using a Becton Dickinson FACScan cytometer.
For cell cycle analysis, cells were fixed in 75% ethanol at −20°C overnight and washed with cold PBS, treated with 100 μg RNase A (Sigma), and stained with 50 μg propidium iodide (Roche).
Measurement of VEGF production was determined by intracellular staining according to the manufacturer’s protocol (BD Biosciences). After monocytes had differentiated into macrophages, cells in 2% FBS melanoma media were incubated for 4 hours in the presence of the indicated concentration of PLX4720 and GolgiPlug. After cells were washed with FACS buffer, intracellular staining was performed with a R-Phycoerythrin-conjugated anti-VEGF mAb according to the manufacturer’s instructions (R&D Systems).
For FACS analysis of peritoneal macrophages, 10 ml of cold PBS was intraperitoneally injected into the mice after they were euthanized. Cells were harvested and the numbers of macrophages were counted with a hemocytometer. An anti-mouse F4/80 (BD Biosciences) was used to analyze the percent of macrophages. All FACS data were analyzed with FlowJo software (TreeStar).
Wang T., Xiao M., Ge Y., Krepler C., Belser E., Lopez-Coral A., Xu X., Zhang G., Azuma R., Liu Q., Liu R., Li L., Amaravadi R.K., Xu W., Karakousis G., Gangadhar T.C., Schuchter L.M., Lieu M., Khare S., Halloran M.B., Herlyn M, & Kaufman R.E. (2015). BRAF Inhibition Stimulates Melanoma-Associated Macrophages to Drive Tumor Growth. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(7), 1652-1664.
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8

Characterizing Macrophage Populations in Humanized Mice

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Anti-human CD45 (Cell Signaling, Danvers, MA) at a dilution of 1:250 and 3, 3-diaminobenzidine (DAB; Vector Labs, CA) was used to stain lung and liver tissues. CD45+ expression was observed and recorded at 10×. Immunohistochemistry staining for anti-human CD206 (Sigma-Aldrich, MO) was performed by the Northwestern Pathology Core Facility to identify presence of human alveolar macrophages in representative lung samples. Staining for mouse-specific macrophages was performed using anti-mouse F4/80 (BD Biosciences) with biotin labelled secondary antibody. Lung tissue from a non-humanized mice strain (C57BL/6J, wildtype adult mice 12 months, provided by Eniko Sajti) was used as a positive control.
Birkett R., Newar J., Sharma A.M., Lin E., Blank L., Swaminathan S., Misharin A, & Mestan K.K. (2023). Development of a novel humanized mouse model to study bronchopulmonary dysplasia. Frontiers in Pediatrics, 11, 1146014.
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