Anti f4 80

Manufactured by BD

Sourced in United States, United Kingdom

Anti-F4/80 is a laboratory reagent used for the detection and identification of mouse macrophages. It is an antibody that specifically binds to the F4/80 antigen, a cell surface glycoprotein expressed on the majority of mature tissue macrophages.

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Lab products found in correlation

Anti cd11b, by BD (24 mentions) Anti cd45, by BD (16 mentions) Anti cd11c, by BD (16 mentions) Anti cd3, by BD (11 mentions) Anti cd4, by BD (11 mentions) Anti ly6g, by BD (9 mentions) Anti ly6c, by BD (7 mentions) Anti cd19, by BD (6 mentions) Anti gr 1, by BD (6 mentions) Anti cd86, by BD (6 mentions) Anti cd8, by BD (5 mentions) Anti b220, by BD (5 mentions) Anti gr 1 antibody, by BD (4 mentions) Anti α sma, by Abcam (4 mentions) Anti cd206, by BD (4 mentions) Anti cd11c, by BioLegend (3 mentions) Anti cd11b clone m1 70, by BD (3 mentions) Anti cd8a, by BD (3 mentions) Anti s100a4, by Abcam (3 mentions) Alexa fluor 488 or 555 conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)

53 protocols using anti f4 80

Histological Analysis of Adipose and Liver Tissues

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Cryostat sections from white adipose tissues (WATs) and liver tissues were prepared. Five micrometer-thick sections were stained with H&E for histological analysis or Oil Red O for the fat droplets accumulation.
For immunohistochemistry (IHC) staining, cryostat sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-F4/80 (BD Pharmingen, San Diego, CA), and anti-Gr-1 antibodies (BD Pharmingen, San Diego, CA), respectively, then were incubated with species matched Alexa dye-labeled or horseradish peroxidase-conjugated secondary antibodies.
For fluorescence double staining, cryostat sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-F4/80 (BD Pharmingen, San Diego, CA), anti-α-SMA (Abcam, Cambridge, UK) antibodies, followed by the staining with Alexa Fluor 488 or 555-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Sections were evaluated under the microscope (DP71, OLYMPUS) for bright-field and fluorescence microscopy.

Hou S., Jiao Y., Yuan Q., Zhai J., Tian T., Sun K., Chen Z., Wu Z, & Zhang J. (2018). S100A4 protects mice from high-fat diet-induced obesity and inflammation. Laboratory investigation; a journal of technical methods and pathology, 98(8).

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Pancreatic Tissue Dissociation and Cell Isolation

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The digestion of the pancreas was performed as described previously (3 (link)). Briefly, pancreatic duct perfusion was performed, after which the pancreas was subsequently digested with 0.25 mg/ml collagenase (Sigma-Aldrich, Pittsburgh, PA, USA) for 30 minutes and with 10 μg/ml trypsin and 10 μg/ml DNase for approximately 20 minutes to obtain a single pancreatic cell population. Flow cytometry was done based on direct fluorescence for mT and CD45 or F4/80, using a pre-incubation with PE-cy7-conjugated anti-CD45 or anti-F4/80 (Becton-Dickinson Biosciences, San Jose, CA, USA), respectively.

Jiang Y., Wiersch J., Wu W., Qian J., Adama M.P., Wu N., Yang W., Chen C., Zhu L., Prasadan K., Gittes G.K, & Xiao X. (2023). Bone-marrow derived cells do not contribute to new beta-cells in the inflamed pancreas. Frontiers in Immunology, 14, 1084056.

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Immunophenotyping of Mouse Immune Cells

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The fluorochrome-conjugated mAb to mouse antigens used for flow cytometry analysis were from eBioscience (VWR, Haasrode, Belgium): anti-CD16/CD32, anti-Ly-6c (PE conjugated; clone HK1.4), anti F4/80 (APC conjugated; clone BM8), anti-Foxp3 (APC conjugated; clone FJK-16s); from Beckton Dickinson (Erembodegem, Belgium): anti-CD3 (PerCp conjugated; clone 145-2C11), anti-CD4 (FITC conjugated; clone RMA4-4), anti-CD8 (Pe-Cy7 conjugated; clone 53-6.7), anti-CD45 (APC-Cy7 conjugated; clone 30-F11), anti-CD25 (PE conjugated; clone 7D4), anti-CD11b (FITC conjugated; clone M1/70); or from Biolegend (Imtec, Antwerpen Belgium): anti-CD11c (Pe/Cy7 conjugated; clone N418), anti-Ly-6G (Brillant violet 421 conjugated; clone 1A8). GK1.5 mAb reportedly do not block binding of RM4-4 antibodies.

Baudoux T., Husson C., De Prez E., Jadot I., Antoine M.H., Nortier J.L, & Hougardy J.M. (2018). CD4+ and CD8+ T Cells Exert Regulatory Properties During Experimental Acute Aristolochic Acid Nephropathy. Scientific Reports, 8, 5334.

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Isolation and Analysis of Lung Immune Cells

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Lungs were perfused with PBS containing 2% fetal calf serum (FCS), excised and finely minced, followed by enzymatic digestion for 30 min at 37°C in PBS containing 150U/ml collagenase type IV and 20 U/ml DNase type I. Lung homogenates were suspended in a 20% Percoll gradient and centrifuged. Pellets were then washed, and red blood cells lysed. Cells were incubated in RPMI 10% FCS containing Golgi Plug/Golgi Stop for 2 hours at 37°C. Cells were then stained for 30 minutes with fluorescence-conjugated antibodies (Biolegend or Becton Dickinson) diluted in PBS 2% FCS: anti-CD45 (AF700-conjugated), anti-Ly6G (FITC-conjugated), anti-CD11b (PerCpCy5.5-conjugated), anti-SiglecF (APCCy7-conjugated), anti-CD11c (FITC-conjugated), anti-MHCII (PerCpCy5.5-conjugated), anti-F4/80 (PeCy7-conjugated), anti-CD4 (FITC-conjugated), anti-CD8 (APCCy7-conjugated), anti-B220 (PerCpCy5.5-conjugated) and anti-PD-L1 (PeCy7-conjugated). Cells were washed and fixed, permeabilized, and stained with PE-conjugated antibodies against pro-IL-1β (Thermofisher) or IL-17A (Biolegend) and analyzed on a BD Aria cell sorter. Flow cytometry analyses were performed using the Diva software.

Hassane M., Rahal Z., Karaoghlanian N., Zhang J., Sinjab A., Wong J.W., Lu W., Scheet P., Lee J.J., Raso M.G., Solis L.M., Fujimoto J., Chami H., Shihadeh A.L, & Kadara H. (2022). Chronic exposure to waterpipe smoke elicits immunomodulatory and carcinogenic effects in the lung. Cancer prevention research (Philadelphia, Pa.), 15(7), 423-434.

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Isolation of Interscapular Brown Adipose Tissue Cells

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Interscapular BAT was dissected from the mice gavaged with Bacteroides or vehicle for 12 weeks and processed for cell isolation as described previously (Hagberg et al., 2018 (link)). Briefly, 100 mg of BAT was minced carefully using scissors for 3 min. The minced tissue was digested in a 37°C thermal shaker (#0003637; TAITEC, Saitama, Japan) for 30 min with Collagenase Type 1 (#LS004196; Worthington Biochemical Corp., Lakewood, NJ) prepared in 10 mL PBS. The samples were then centrifuged for 2 min at 20 × g, and the pellets (stromal vascular fraction) were washed 2 times with 10 mL PBS containing 2% bovine serum albumin and incubated with an anti-CD16/CD32 antibody (#553142; BD Biosciences) to block Fc receptors. This was followed by staining with the following antibodies: anti-CD45 (#557659; BD Biosciences), anti-F4/80 (#565411; BD Biosciences), anti-CD11b (#563402; BD Biosciences), and anti-Ly6G (#560602; BD Biosciences). Samples incubated with the isotype-matched antibodies were used as controls. Flow cytometric analysis was performed on an Attune acoustic focusing cytometer (Life Technologies, Grand Island, NY) and by using FlowJo software (Tree Star, Inc., Ashland, OR).

Yoshida N., Yamashita T., Osone T., Hosooka T., Shinohara M., Kitahama S., Sasaki K., Sasaki D., Yoneshiro T., Suzuki T., Emoto T., Saito Y., Ozawa G., Hirota Y., Kitaura Y., Shimomura Y., Okamatsu-Ogura Y., Saito M., Kondo A., Kajimura S., Inagaki T., Ogawa W., Yamada T, & Hirata K.I. (2021). Bacteroides spp. promotes branched-chain amino acid catabolism in brown fat and inhibits obesity. iScience, 24(11), 103342.

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Peritoneal Immune Cell Profiling in CLP

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Peritoneal lavage was harvested from mice at different time points before and after CLP. Peritoneal exudate cells were incubated with anti-CD11b (BD Pharmingen), anti-CD11c (BioLegend), anti-F4/80 (BD Pharmingen), and anti-Gr1 (BioLegend) monoclonal antibodies (mAbs) conjugated with FITC, PE, PerCP, or APC. FITC-, PE-, PerCP-, and APC-conjugated anti-mouse isotype-matched mAbs (BD Pharmingen; BioLegend) were used as negative controls. Subpopulations of macrophages (CD11b⁺F4/80⁻CD11c^lo) and PMNs (CD11b⁺F4/80⁻Gr1^hi) in the peritoneal cavity were detected by FACScan analysis (BD Biosciences).

Huang J., Ita M., Zhou H., Zhao H., Hassan F., Bai Z., O’Leary D.P., Li Y., Redmond H.P., Wang J.H, & Wang J. (2022). Autophagy induced by taurolidine protects against polymicrobial sepsis by promoting both host resistance and disease tolerance. Proceedings of the National Academy of Sciences of the United States of America, 119(19), e2121244119.

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Multiparametric Flow Cytometry Analysis of Immune Cell Subsets

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Surface and intracellular staining were performed as previously described [28 (link)]. Single immune cell populations from the spleen, lymph nodes or tumors were separated with a BD FACSAria II Cell Sorter. Flow cytometric analyses were performed with Flowjo (Tree Star). The following antibodies were used for cell staining: anti-CD3 (clone 145-2C11), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD49b (clone DX5), anti-CD11b(clone M1/70), anti-CD11c (clone HL3), anti-CD19 (clone 1D3), anti-CD25 (clone PC61), anti-CD69 (clone H1.2F3), anti-CD62L (MEL-14), anti-CD44 (clone IM7), anti-Foxp3 (clone FJK-16s), anti-Granzyme B (clone GB11), anti-CCR4 (clone 2G12), anti-CCR5 (clone HM-CCR5), anti-CXCR3 (clone CXCR3-173), NK1.1(clone PK136), anti-F4/80 (clone BM8), anti-Gr-1 (clone RB6-8C5), anti-interferon-γ (IFN-γ, clone XMG1.2), and anti-NK1.1 (clone PK136), CD4 blocking mAb (clone GK1.5), and CD8 blocking mAb (clone 53-6.7).
For detection of phosphorylated S6 proteins, cells from LNs cultured with PMA (10ng/ml) and Ionomycin (500ng/ml) at designated times were immediately fixed with phosflow Lyse/Fix buffer (BD Biosciences) and permeabilized by Phosflow Perm buffer (BD Biosciences). Cells were stained with the Alex488 conjugated antibody for S6P (Ser235,236) (D57.2. 2E; Cell Signaling Technology)

Rao E., Zhang Y., Zhu G., Hao J., Persson X.M., Egilmez N.K., Suttles J, & Li B. (2015). Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death. Oncotarget, 6(10), 7944-7958.

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Quantifying Macrophage Populations by Flow Cytometry

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Cells (3×10⁵cells) were incubated first with mouse Fc Receptor Block (2.4G2; BioXcell, West Lebanon, NH, USA) to inhibit non-specific binding of antibodies. After washing, cells were stained with anti-F4/80, anti-CD11b, anti-CD11c and anti-Gr-1' antibodies (BD Pharmingen). Numbers of positive cells were quantified by flow cytometry (FACSCanto flow cytometer, BD Biosciences, San Jose, CA). BMDMs were categorized by F4/80⁺CD11b⁺CD11c⁻Gr-1 cells [51 , 52 (link)]. Data was collected on a FACS Canto flow cytometer and analysed with FlowJo software (TreeStar, Ashland, OR, USA).

Zhou H., Zhang J., Eyers F., Xiang Y., Herbert C., Tay H.L., Foster P.S, & Yang M. (2016). Identification of the microRNA networks contributing to macrophage differentiation and function. Oncotarget, 7(20), 28806-28820.

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Murine Bone Marrow-Derived Macrophage Isolation

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Murine BMDMs were generated by the following procedure. Marrow from both femurs and tibiae was harvested in DMEM/F-12 (Gibco) supplemented with 10% heat-inactivated FBS (HyClone, Thermo Fisher Scientific) and flushed through a syringe with a 20-gauge needle. The dispersed cells were then seeded in Petri dishes (100 × 15 mm) containing 10 ml DMEM/F-12 supplemented with 2 mM l-glutamine (Gibco), 10% FBS, 2% Hepes (Life Technologies), 1 mM sodium pyruvate (Gibco), 25 µg/ml gentamicin (Gibco), and 20% of L929-conditioned media. After 3 d of incubation at 37°C with 5% CO₂, fresh medium containing L929-conditioned media without gentamicin was added. On day 6, macrophages were detached by the addition of cold Dulbecco’s PBS (DPBS). The resulting cells were shown to be ∼95% pure based on extracellular double staining with anti-CD11b (eBioscience) and anti-F4/80 (BD Biosciences) macrophage markers by flow cytometric analysis.

Amaral E.P., Costa D.L., Namasivayam S., Riteau N., Kamenyeva O., Mittereder L., Mayer-Barber K.D., Andrade B.B, & Sher A. (2019). A major role for ferroptosis in Mycobacterium tuberculosis–induced cell death and tissue necrosis. The Journal of Experimental Medicine, 216(3), 556-570.

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Multiparametric Flow Cytometry of Tumor-Infiltrating Immune Cells

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Mouse blood was retroorbitally collected in heparinized capillary tubes followed by RBC lysis (BioLegend). Mouse tumors were collected after sacrifice and dissociated using 30% collagenase digestion (30 mins, 37°C). Single cell suspensions in 5% FBS in PBS by passing through 40 μm cell strainer were assessed for viability with Trypan Blue and manually counted, then incubated with Fc receptor blocking solution followed by fluorophore-conjugated antibodies.
For cell surface staining, fluorescence-labeled mouse antibodies (including anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD11b, anti-Ly6C, anti-Ly6G, anti-F4/80, anti-CD11c, anti-CD80, anti-CD86, anti-MHCII purchased from BD Biosciences, BioLegend or eBioscience) were added to samples for 30 min at 4 °C in the dark. For further intracellular staining, cells were washed with PBS supplemented with 5% FBS, permeabilized with Permeabilization Buffer (BD Bioscience) and stained with fluorescence-labeled mouse antibodies (including anti-IFNγ, anti-GzmB, and anti-TNFa purchased from BD Biosciences or BioLegend) for 30 min at 4 °C in the dark. Cells were washed and resuspended in 5% FBS in PBS. Flow cytometry was performed on an LSRII (BD Biosciences), and data analyzed using FlowJo Version X (Tree Star).

Ye J., Mills B.N., Zhao T., Han B.J., Murphy J.D., Patel A.P., Johnston C.J., Lord E.M., Belt B.A., Linehan D.C, & Gerber S.A. (2019). Assessing the magnitude of immunogenic cell death following chemotherapy and irradiation reveals a new strategy to treat pancreatic cancer. Cancer immunology research, 8(1), 94-107.

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