F4 80 af647

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F4/80-AF647 is a fluorochrome-conjugated antibody that binds to the F4/80 antigen, which is expressed on the surface of mature mouse macrophages. This antibody can be used in flow cytometry applications to detect and quantify macrophage populations in mouse samples.

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F4/80 (349 citations) AF647 anti-mouse F4/80 (3 citations) Anti-F4/80-AF647 (2 citations)

6 protocols using f4 80 af647

Immunophenotyping of Myeloid Cells

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Cells were harvested from the BM, SP, and FL of a WT mouse, stained in FACS buffer with FVD780 (eBioscience), F4/80-AF647, CD169-PE, and VCAM-AF488 (Biolegend), and analyzed on a FACSCanto flow cytometer (BD). The data were analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA).

Seu K.G., Papoin J., Fessler R., Hom J., Huang G., Mohandas N., Blanc L, & Kalfa T.A. (2017). Unraveling Macrophage Heterogeneity in Erythroblastic Islands. Frontiers in Immunology, 8, 1140.

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Tumor Immune Cell Profiling Protocol

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Tumors were minced and digested by using the mouse Tumor Dissociation Kit (Miltenyi Biotech), washed with FACS buffer (PBS containing 2% FBS, 2 mM EDTA and 0.02% sodium azide), and filtered through a 70 -µm filter (BD Biosciences). The obtained single-cell suspension was stained with LIVE/DEAD Fixable Blue Dead Cell Stain kit (Thermo Fisher Scientific), blocked with anti-CD16/32 (BD Biosciences; #553142; clone 2.4G2; 1:1000), stained with fluorochrome-labeled antibodies, and analyzed using a LSR Fortessa (BD Biosciences) and FlowJo software (Tree Star Inc).
The following antibodies were purchased from BD Biosciences: CD3-BUV737 (#564380; clone 17A2; 1:400), CD8a-BB515 (#564422; clone 53-6.7; 1:400), CD44-APC-Cy7 (#560568; clone IM7; 1:1000), CD274-BV711 (#563369; clone MIH5; 1:200), CD11b-FITC (#557672; clone M1/70; 1:800), CD45-BV605 (#563053; clone 30F11; 1:400). The following antibodies were purchased from Biolegend: CD4-BV510 (#100449; clone GK1.5; 1:400), CD62L-BV785 (#104440; clone MEL-14; 1:200), CD161-PE (#108707; clone PK136; 1:400), CD11c-PE (#117308; clone N418; 1:200), F4/80-AF647 (#123122; clone BM8; 1:2000).

Galvani E., Mundra P.A., Valpione S., Garcia-Martinez P., Smith M., Greenall J., Thakur R., Helmink B., Andrews M.C., Boon L., Chester C., Gremel G., Hogan K., Mandal A., Zeng K., Banyard A., Ashton G., Cook M., Lorigan P., Wargo J.A., Dhomen N, & Marais R. (2020). Stroma remodeling and reduced cell division define durable response to PD-1 blockade in melanoma. Nature Communications, 11, 853.

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Multiparametric Flow Cytometry of Tumor Cells

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Single-cell suspensions of tumors were prepared, and flow cytometry was carried out as mentioned earlier [45 (link), 46 (link)]. Cells were stained with the following antibodies obtained from BioLegend: CD16/32 (clone 93, catalog No. 103132), Zombie UV™ Fixable Viability Kit (catalog No. 100752), CD45-PerCP/Cy5.5 (clone 30-F11, catalog No. 103132), CD45-APC/Cy7 (clone 30-F11, catalog No. 103116), CD11b-FITC (clone M1/70, catalog No. 101206), SIRPα-PE (clone BM8, catalog No. 123122), Gr-1-PerCP/Cy5.5 (clone RB6-8C5, catalog No. 108428), F4/80-AF647 (clone N418, catalog No. 117318),CD11c-PE/Cy7 (clone N418, catalog No. 117318), I-A/I-E-AF700 (clone M5/114.15.2, catalog No. 107622), CD86-BV421 (clone GL-1, catalog No. 105032), CD206-BV605 (clone C068C2, catalog No. 141721), CD8a-AF700 (clone 53-6.7, catalog No. 100730), CD8-BV510 (clone 53-6.7, catalog No. 100752), TNF-α-BV421 (clone MP6-XT22, catalog No. 506328), IFN-γ-PE (clone XMG1.2, catalog No. 505808), CD39-PE/Cy7 (clone Duha59, catalog No. 143806), Tim-3-APC/Fire™ 750 (clone RMT3-23, catalog No. 119738), PD-1-BV605 (clone 29 F.1A12, catalog No. 135220). The next antibodies were obtained through eBioscience: Mouse Regulatory T Cell Staining Kit #2 (clone FJK-16s, catalog No. 88-8118-40). FlowJo program (Tree Star Inc.) was utilized to examine the findings that were acquired through a BD LSRFortessa Flow Cytometer.

Ji K., Zhang Y., Jiang S., Sun L., Zhang B., Hu D., Wang J., Zhao L., Wang P, & Tao Z. (2023). SIRPα blockade improves the antitumor immunity of radiotherapy in colorectal cancer. Cell Death Discovery, 9, 180.

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Immune Cell Profiling in Liver

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Immune cells were isolated from the liver using two-step collagenase perfusion with liver perfusion medium (Gibco) and liver digest medium (Gibco) and filtered through a 100-µm cell strainer. Hepatocytes were removed by centrifugation at 50 g. After lysing red blood cells (Sigma-Aldrich), cells were incubated with FC-Block (BD) and the following antibodies against immune cell surface markers: F4/80-AF647, CD45-PerCP, and CD45R-APC-Cy7 (BioLegend); CD3-AF700 (eBioscience); and CD4-PE-Cy7, CD8-PE-Cy5, NK-1.1-PE, and Ly-6G/Ly-6C-PerCP-Cy5.5 (BD). Cells were fixed in 2% PFA. Data were acquired on a BD LSRII Fortessa and analyzed using FlowJo 9.5.3. Live cells were gated for CD45⁺, and at least 10,000 individual cells were collected. The different immune cell populations in the CD45⁺ population were gated as follows: T cells, CD3⁺, NK1.1⁻; NK cells, CD3⁻, NK1.1⁺; NKT cells, CD3⁺, NK1.1⁺; CD4⁺ T cells, CD4⁺, CD8⁻; CD8⁺ T cells, CD4⁻, CD8⁺; macrophages and monocytes, F4/80⁺; B cells, CD45R⁺ (B220⁺); and granulocytes, Gr1⁺ (Ly-6G/Ly-6C).

Bakiri L., Hamacher R., Graña O., Guío-Carrión A., Campos-Olivas R., Martinez L., Dienes H.P., Thomsen M.K., Hasenfuss S.C, & Wagner E.F. (2017). Liver carcinogenesis by FOS-dependent inflammation and cholesterol dysregulation. The Journal of Experimental Medicine, 214(5), 1387-1409.

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Quantifying Tumor-Associated Macrophages in Frozen Samples

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Tumors were resected from tumor-bearing mice and cut in half. The tissue was incubated in fixation solution [4% paraformaldehyde/20% sucrose] for 1 hour at room temperature and 5 hours at 4ºC. Tissue was frozen on dry ice in optimal cutting temperature (OCT) medium and stored at −80ºC. To obtain tissue slides, the tissue was sectioned to 0.08 μm with a Leica 3000 cryostat. After sectioning, slides were dried at 37ºC and permeabilized for 20 minutes at room temperature with 1 mM Triton-x100 in TBST before being blocked for 1 hour at room temperature with 50% SEA Block in PBS. Tissue was stained with antibodies to F4/80–AF647 at 1:100 dilution (BioLegend), GFP-AF488 at 1:200 dilution (Abcam) and DAPI at 1:10,000 dilution in 5% SEA Block. Images were taken using a fluorescent microscope. Three-dimensional rendering was performed using images from a confocal microscope at the Harvard Medical School MicRoN Imaging Core Facility.

Roehle K., Qiang L., Ventre K., Heid D., Ali L.R., Lenehan P., Heckler M., Crowley S.J., Stump C.T., Ro G., Godicelj A., Bhuiyan A.M., Yang A., del Rey M.Q., Biary T., Luoma A.M., Bruck P.T., Tegethoff J.F., Nopper S.L., Li J., Byrne K.T., Pelletier M., Wucherpfennig K.W., Stanger B.Z., Akin J.J., Mancias J.D., Agudo J., Dougan M, & Dougan S.K. (2021). cIAP1/2 antagonism eliminates MHC class I negative tumors through T cell-dependent reprogramming of mononuclear phagocytes. Science translational medicine, 13(594), eabf5058.

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Multicolor Flow Cytometry Profiling

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100 µL of the above cell suspensions was transferred to a round bottom 96-well plate and centrifuged (3000 rpm, 1 min, 4 °C). Cell were blocked at 4 °C for 15 min using 24G2 blocking antibody (Preclin Biosystems AG) at 800 µg/mL final concentration. Cells were washed by centrifugation and resuspension with PBS/0.2% BSA. Cell were then centrifuged (3000 rpm, 1 min, 4 °C) and resuspended in 50 µl of staining solution containing appropriate primary antibodies (CD11b-PerCp-Cy5.5; CD11c-APC-Cy7; MHCII-AF700; F4/80-AF647; CD40-PE/Cy5; CD86-PE; CD80-PE/Cy7; CD4-Pacific blue; CD8-APC-Cy7; CD25-AF700; CD44-PE; all Biolegend) for 30 min at 4 °C. After two washes with PBS/0.2% BSA and one wash in PBS cells were fixed in 50 µL of BD FACS lysing solution for 15 min at 4 °C. Cells were then washed twice in PBS/0.2% BSA and resuspended in 100 µL of PBS/0.2% BSA. For intracellular staining cells were washed once more in 0.5% saponin. Cells were then incubated with primary antibody (FoxP3-AF647, Biolegend) overnight at 4 °C in 0.5% saponin. Cells were then washed once with 0.5% saponin and twice with PBS/0.2% BSA before being resuspended in 100 µL of PBS/0.2% BSA for flow cytometry analysis.

Raftis E.J., Delday M.I., Cowie P., McCluskey S.M., Singh M.D., Ettorre A, & Mulder I.E. (2018). Bifidobacterium breve MRx0004 protects against airway inflammation in a severe asthma model by suppressing both neutrophil and eosinophil lung infiltration. Scientific Reports, 8, 12024.

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