F4 80 bv650

Manufactured by BioLegend

F4/80 BV650 is a fluorescently-labeled antibody product manufactured by BioLegend. It is designed for the detection and analysis of F4/80-expressing cells, which are commonly associated with macrophages, using flow cytometry.

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F4/80 (349 citations)

4 protocols using f4 80 bv650

Investigating Ovarian Cancer Immunotherapy

Cited 1 time

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Conventional C57BL/6 mice were obtained from Envigo and housed at the pathogen-free animal facility of the Ludwig Institute in Epalinges (license 2797.1g), under approved protocols. Mice (n=15 per group) were injected i.p. with 0.5 million Tp53^−/−Brca1^−/− ID8 ovarian cancer cells (a kind gift of Dr. Ian McNeish, Imperial College London (Walton et al., 2017 (link)) expressing luciferase (Bruand et al., 2021 ) and evaluated weekly for the luciferase signal by IVIS Lumina (Perkin Elmer). Mice were treated with 100 μg/mouse of αPD-1 (RMP1–14, BioX Cell), αCTLA-4 (9D9, BioX Cell), CD40L (FGK45), or αCD28 antibody (E18, BioLegend), 3 times/week for 3 weeks, except for CD40L (twice/week, 2 weeks), starting 5 days after tumor injection. At the end point, mice were euthanized, and blood and tumors were collected. Tumors were minced, dissociated and stained for CD45.2 BUV737 (104), CD83 BV711 (Michel.19), I-a/I-E FITC (2G9), Gr1 FITC (RB6–8C5) from BD, PD-1 BV510 (29F.1A12), PD-L1 BV785 (10F.9G2), CD80 BV421 (16–10A1), CD86 APC-A780 (GL-1), CD4 BV711 (RM4–5), F4/80 BV650 (BM8), CD11b BV605 (M1/70), Ki-67 PEDazzle (16A8), CD103 PE (2E7), CD137 APC (17B5) from BioLegend, and PD-L2 PerCP Cy5.5 (122), CD8a APCeFluo780 (53.6.7), and CD11c APC (N4/18) from ThermoFisher Scientific.

Duraiswamy J., Turrini R., Minasyan A., Barras D., Crespo I., Grimm A.J., Casado J., Genolet R., Benedetti F., Wicky A., Ioannidou K., Castro W., Neal C., Moriot A., Renaud-Tissot S., Anstett V., Fahr N., Tanyi J.L., Eiva M.A., Jacobson C.A., Montone K.T., Wulff Westergaard M.C., Svane I.M., Kandalaft L.E., Delorenzi M., Sorger P.K., Färkkilä A., Michielin O., Zoete V., Carmona S.J., Foukas P.G., Powell DJ J.r., Rusakiewicz S., Doucey M.A., Laniti D.D, & Coukos G. (2021). Myeloid Antigen-Presenting Cell Niches Sustain Antitumor T Cells and License PD-1 Blockade via CD28 Costimulation. Cancer cell, 39(12), 1623-1642.e20.

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Immune cell profiling of quadriceps muscle

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Perfused ipsilateral quadriceps muscles were minced and digested in RPMI supplemented with 10% HI-FBS, HEPES pH 7.3, 100 U/ml penicillin and streptomycin, collagenase (2.5 mg/ml; Sigma), and DNase I (30 μg/ml; Roche) in a total of 5 ml at 37°C for 1.5 h. Digested tissue was pressed through a 70 μm cell strainer, and resuspended in RPMI supplemented with 10% HI-FBS and penicillin and streptomycin. Cells were counted using precision count beads (BioLegend). Single cell suspensions were blocked for FcγR binding (BioLegend; clone S17011E) and stained with the following antibodies: CD3 BV421 (BioLegend; clone 145-2C11), CD4 FITC (BioLegend; clone RM4-5), CD8α APC (BioLegend; clone 53–6.3), NK1.1 PE (BioLegend; clone PK136), CD45 BUV395 (BD Biosciences; clone 30-F11), CD19 BV605 (BioLegend; clone 6D5), CD11b PerCP-Cy5.5 (BioLegend; clone M1/70), Ly6C Pacific Blue (BioLegend; clone HK1.4), Ly6G phycoerythrin (PE)-Cy7 (BioLegend; clone 1A8), MHC class II Alexa Fluor 700 (BioLegend; clone M5/114.15.2), Ly6B FITC (BioLegend; clone 7/4), and F4/80 BV650 (BioLegend; clone BM8). Viability was determined by exclusion of a fixable viability dye (eBiosciences; e506). Samples were run on a BD LSRFortessa X-20 flow cytometer and analyzed using FlowJo version 10 (FlowJo, LLC).

Fox J.M., Huang L., Tahan S., Powell L.A., Crowe JE J.r., Wang D, & Diamond M.S. (2020). A cross-reactive antibody protects against Ross River virus musculoskeletal disease despite rapid neutralization escape in mice. PLoS Pathogens, 16(8), e1008743.

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Comprehensive Immune Cell Profiling

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The adipose SVCs and splenocytes were incubated for 10 minutes with CD16/32 antibody (FC block) (eBioscience) and then stained with other fluorochrome-conjugated antibodies for 20 minutes at 4℃. All cells were stained with antibodies against CD45-PE/Cy7 (eBioscience), CD4-BV500 (BD Biosciences), CD8-APC/Cy7 (Biolegend), CD19-PE (Biolegend), NK1.1-BV421 (Biolegend), NKp46-PE/Dazzle594 (Biolegend), CD11b-BV786 (Biolegend), CD11c-APC (Biolegend), CD3-BV711 (Biolegend), and F4/80-BV650 (Biolegend). 7AAD (Invitrogen) was used for live/dead staining. The stained cells were washed and analyzed with an LSRFortessa flow cytometer (BD Biosciences). NK cells were defined as CD45⁺ F4/80⁻ CD19⁻ CD3⁻ NK1.1⁺ in lymphocyte gating. The NKp46⁺ and GFP⁺ cells were then gated. ATMs were defined as CD45⁺ CD19⁻ CD3⁻ NK1.1⁻ F4/80⁺ CD11b⁺. Total cell numbers were counted with a hemocytometer and normalized according to the tissue weight (cells/g). Flow cytometric data were analyzed by using the FlowJo software (Flow Jo).

Nathalie G., Bonamichi B.D., Kim J., Jeong J., Kang H., Hartland E.R., Eveline E, & Lee J. (2023). NK cell-activating receptor NKp46 does not participate in the development of obesity-induced inflammation and insulin resistance. Molecules and Cells, 47(3), 100007.

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Multicolor Flow Cytometry Assay for Immune Profiling

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Tumor cell suspensions (4 × 10⁶) prepared by mechanical and enzymatic dissociation^{16 (link)} were stained in 96-wells round bottom plates with live/dead staining (Blue fluorescent reactive dye, #L34962 Invitrogen) during 20 min at room temperature. For flow-cytometry analysis and sorting, Fc receptors were blocked with anti-FcR (anti-CD16 and CD32, at 5 µg/ml, Biolegend #101339). After two washes in PBS 2% FCS, cells were stained with the following antibodies (all used at 1:100): anti-CD11b-BV421 (#562605), CD64-APC (#558539), CD11c-PeCy7 (#557401), TCRβ-BV605 (#562840) and CD4-BV711 (#563050) all purchased from BD Pharmingen; anti-CD45-AF700 (#103128), Ly6C-APCCy7 (#128025), Ly6G-BV510 (#127633), F4/80 BV650 (#123149), CD206-PE (#141706), IA/IE-BV785 (#107645) all purchased from Biolegend, and CD8-PerCPef710 (#46-0081-82) purchased from eBioscience. After washing, cells were fixed in 1% PFA, stored at 4 °C, and acquired the next day on LSR II or FORTESSA (BD Bioscience). For detection of phosphorylated proteins, cell suspensions were stimulated 3 h with DMXAA 250 µg/ml, fixed immediately in PFA 4%, permeabilized with frozen methanol 90%, stained overnight with 1:100 pTBK1-PE (#13498) antibodies (purchased from Cell signaling), then washed and further stained for multicolor flow cytometry.

Guerin M.V., Regnier F., Feuillet V., Vimeux L., Weiss J.M., Bismuth G., Altan-Bonnet G., Guilbert T., Thoreau M., Finisguerra V., Donnadieu E., Trautmann A, & Bercovici N. (2019). TGFβ blocks IFNα/β release and tumor rejection in spontaneous mammary tumors. Nature Communications, 10, 4131.

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