Anti mouse f4 80 clone bm8

Manufactured by BioLegend

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Anti-mouse F4/80 (clone BM8) is an antibody that binds to the F4/80 antigen expressed on the surface of mouse macrophages and microglia. It can be used for the identification and isolation of these cell types.

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F4/80 (349 citations) Anti-F4/80 (96 citations) F4/80 (BM8, (56 citations) F4/80 (clone BM8, (35 citations) Anti-F4/80 (clone BM8, (26 citations) Anti-mouse F4/80 (25 citations) Clone BM8 (20 citations) Anti-F4/80 (BM8, (17 citations) PE‐Cy7‐anti‐mouse F4/80 (clone BM8, (4 citations) APC anti-mouse F4/80 Clone BM8 (3 citations)

9 protocols using anti mouse f4 80 clone bm8

Macrophage Surface Marker Analysis

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Macrophages were blocked with 1% BSA in PBS for 15 min prior to antibody staining, or with Human TruStain FcX™ (Fc Receptor Blocking Solution, Biolegend). For detection of cell surface markers, antibodies were incubated with samples containing 2 × 10⁵ cells for 30 min. The following antibodies were used: anti-human CD86 (clone REA968) (Miltenyi Biotec), anti-human HLA-DR (clone L243), anti-human CD80 (clone 2D10), anti-mouse CD80 (clone 16-10A1), and anti-mouse F4/80 (clone BM8) from Biolegend. Following incubation, samples were washed and resuspended in PBS and 10,000–50,000 events recorded. All samples were acquired on a LSR II (BD Biosciences) flow cytometer and data was analysed using FlowJo software, version 10.

Drehmer D., Mesquita Luiz J.P., Hernandez C.A., Alves-Filho J.C., Hussell T., Townsend P.A, & Moncada S. (2022). Nitric oxide favours tumour-promoting inflammation through mitochondria-dependent and -independent actions on macrophages. Redox Biology, 54, 102350.

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Immunoblotting and Flow Cytometry Protocols

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For immunoblotting, these antibodies are used: rabbit anti-STAT1 (CST, #14994), rabbit anti–phospho-STAT1 (CST, #7649), rabbit anti-EZH2 (CST, #5246), mouse anti–MHC-I class I (Santa Cruz Biotechnology, #sc-55582), rabbit anti-IFNGR1 (Millipore, #MABF753), and mouse anti–β-actin (Sigma-Aldrich, #A5441).
For flow cytometry, the following fluorochrome-conjugated antibodies are used: anti-mouse H-2K^b/H-2D^b (clone 28-8-6, BioLegend), anti-mouse H-2Kb bound to SIINFEKL (clone 25-D1.16, BioLegend), anti-mouse CD3 (clone 17A2, BioLegend), anti-mouse CD28 (clone 37.51, BioLegend), anti-mouse granzyme B (clone GB11, BioLegend), anti-mouse B220 (clone RA3-6B2, BioLegend), anti-mouse CD49b (clone DX5, BioLegend), anti-mouse Gr-1 (clone RB6-8C5, BioLegend), anti-mouse MHC-II (clone M5/114.15.2, BioLegend), anti-mouse F4/80 (clone BM8, BioLegend), anti-mouse IFN-γ (XMG1.2, eBioscience), anti-mouse CD11b (M1/70, eBioscience), anti-mouse NK1.1 (clone PK136, BD), anti-mouse CD103 (clone M290, BD), anti-mouse CD206 (clone MR5D3, BD), anti-mouse CD24 (clone M1/69, BD), anti-mouse CD4 (clone GK1.5, BD), anti-mouse CD45 (clone 30-F11, BD), anti-mouse CD8a (clone 53-6.7, BD), anti-mouse Foxp3 (clone MF23, BD), anti-mouse γδ TCR (clone GL3, BD), anti-human CD3 (SK7, BioLegend), anti-human CD4 (SK3, BioLegend), anti-human CD8 (SK1, BioLegend), and anti-human 137(4-1BB) (4B4-1, BioLegend).

Guo W., Wang Y., Yang M., Wang Z., Wang Y., Chaurasia S., Wu Z., Zhang M., Yadav G.S., Rathod S., Concha-Benavente F., Fernandez C., Li S., Xie W., Ferris R.L., Kammula U.S., Lu B, & Yang D. (2021). LincRNA-immunity landscape analysis identifies EPIC1 as a regulator of tumor immune evasion and immunotherapy resistance. Science Advances, 7(7), eabb3555.

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Multiparameter Flow Cytometry Analysis

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Anti-mouse CD47 (clone miap301; BioLegend), anti-human CD47 (clone B6H12; BD Biosciences), anti-mouse F4/80 (clone BM8; BioLegend), anti-mouse/human CD11b (clone M1/70; BioLegend), anti-mouse MHC-II (clone M5/114.15.2; BioLegend), anti-mouse CD206 (clone C068C2; BioLegend), anti-mouse Sirpα (clone P84; BioLegend) and anti-human CD206 (clone 15-2; BioLegend) were used for FACS analyses. Antibodies were phycoerythrin (PE), PE Cy7, APC, APC Cy7, Alexa Fluor 700, BV787, or BV605 conjugated, or fluorophore-conjugated secondary antibodies were used. Sytox blue was used to exclude dead cells. Annexin V (BD Biosciences) and 7-aminoactinomycin D (7-AAD; ThermoFisher) were used to evaluate cell viability of macrophages on cabazitaxel treatment. Flow cytometry was performed using the BD LSRFortessa cell analyzers.

Cao X., Li B., Chen J., Dang J., Chen S., Gunes E.G., Xu B., Tian L., Muend S., Raoof M., Querfeld C., Yu J., Rosen S.T., Wang Y, & Feng M. (2021). Effect of cabazitaxel on macrophages improves CD47-targeted immunotherapy for triple-negative breast cancer. Journal for Immunotherapy of Cancer, 9(3), e002022.

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Tumor Immune Profiling by Flow Cytometry

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Tumor lysates were prepared by mincing the tumor in DMEM (Sigma) and incubating in collagenase (Roche) at 37°C for 1 h. After washing in PBS, the cells were filtered through 70‐μm filters (BD Biosciences). 5 × 10 cells were re‐suspended in HBSS (Hank's balanced salt solution, Lonza) supplemented with 0.5% BSA (Sigma). Staining was performed at 4°C for 20 min, with the following antibodies: anti‐mouse CD45 (clone 30‐F11, Biolegend San Diego, CA, USA), anti‐mouse F4/80 (clone BM8, Biolegend San Diego, CA, USA), anti‐mouse cd11c (clone N418, Biolegend San Diego, CA), anti‐mouse I‐A/I‐E (clone M5/114.15.2, Biolegend San Diego, CA, USA); anti‐mouse TNFα (clone MP6‐XT22, BD Pharmingen), anti‐mouse Ly6C (clone HK1.4, eBioscience; San Diego, CA, USA); anti‐mouse NOS2 (clone 6/iNOS/NOS Type II, BD biosciences, San Diego, CA, USA) and mouse IgG2a K (BD biosciences, San Diego, CA, USA). For intracellular staining, Cytofix/Cytoperm and Permwash staining kit (BD Pharmingen) were used. Cells were detected using the BD FACS Canto II cytofluorimeter and analyzed with FlowJo software.

Comunanza V., Corà D., Orso F., Consonni F.M., Middonti E., Di Nicolantonio F., Buzdin A., Sica A., Medico E., Sangiolo D., Taverna D, & Bussolino F. (2016). VEGF blockade enhances the antitumor effect of BRAFV 600E inhibition. EMBO Molecular Medicine, 9(2), 219-237.

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Multiparametric Flow Cytometry Analyses

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Anti-mouse F4/80 (clone BM8, BioLegend), anti-mouse/human CD11b (clone M1/70, BioLegend), anti-mouse MHCII (clone M5/114.15.2, BioLegend), anti-mouse iNOS (clone W16030C, BioLegend), anti-mouse CD206 (clone C068C2, BioLegend), and anti-mouse CSF1R (clone AFS98, BioLegend) were used for FACS analyses. The antibodies were PE-, PE-Cy7–, allophycocyanin (APC)–, APC Cy7–, or Alexa Fluor 700–conjugated, or fluorophore-conjugated secondary antibodies were used. Sytox blue or Zombie Violet was used to exclude dead cells. Flow cytometry was performed using the BD LSRFortessa cell analyzers.

Cao X., Chen J., Li B., Dang J., Zhang W., Zhong X., Wang C., Raoof M., Sun Z., Yu J., Fakih M.G, & Feng M. (2022). Promoting antibody-dependent cellular phagocytosis for effective macrophage-based cancer immunotherapy. Science Advances, 8(11), eabl9171.

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Detailed Immune Cell Profiling

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Phenotype analysis was performed with staining performed at 4 °C for 20 min with the following antibodies: anti‐mouse CD45 (clone 30‐F11; Biolegend, San Diego, CA, USA), anti‐mouse F4/80 (clone BM8; Biolegend), anti‐mouse CD11c (clone N418; Biolegend), anti‐mouse Ly6C (clone HK1.4; Thermo Fisher Scientific); anti‐CD11b (clone M1/70; Biolegend, San Diego, CA, USA), anti‐CD206 (clone C068C2; Biolegend), anti‐Ly6G (clone 1A8; Biolegend), anti‐NK1.1 (clone PK136; Biolegend), anti‐CD314 (clone C7; Biolegend), anti‐CD4 (clone GK1.5; BD Pharmigen, San Jose, CA, USA), anti CD8 (clone 53‐6.7; BD Pharmigen); antiPD‐1 (clone 29F.1A12; Biolegend), anti‐PD‐L1 (clone 10F.9G2; Biolegend). Cells were detected using the Cyan ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed with the Summit 4.3 software (Beckman Coulter). Quadrants were set based on isotype control antibody, and cells were gated among total DAPI⁻ cells.

Comunanza V., Gigliotti C., Lamba S., Doronzo G., Vallariello E., Martin V., Isella C., Medico E., Bardelli A., Sangiolo D., Di Nicolantonio F, & Bussolino F. (2023). Dual VEGFA/BRAF targeting boosts PD‐1 blockade in melanoma through GM‐CSF‐mediated infiltration of M1 macrophages. Molecular Oncology, 17(8), 1474-1491.

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Tumor-Associated Immune Cell Profiling

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Mice with tumour formation were euthanised by CO₂ gas, and their spleens, femurs, tibias and tumours were collected. Cells were stained with the following antibodies (BioLegend, San Diego, USA) for 30 min at 4 °C: anti-mouse CD3 (clone 145-2C11), anti-mouse CD4 (clone RM4-5), anti-mouse CD8a (clone 53-6.7), anti-mouse CD45 (clone 30-F11), anti-mouse Gr-1 (clone RB6-8C5), anti-mouse CD11b (clone M1/70), anti-mouse Ly6G (clone 1A8), anti-mouse Ly6C (clone AL-21), anti-mouse F4/80 (clone BM8) and anti-mouse CD11c (clone N418). Non-viable cells were stained with 7-Amino-actinomycin D (AAD) staining solution or DAPI solution, or Zombie Aqua^TM and gated out. Matched isotype antibodies were used as controls. Data were acquired using a MACS Quant (Miltenyi Biotec, Bergisch Galdbach, Germany) and analysed by MACS Quantify (Miltenyi Biotec).

Horikawa N., Abiko K., Matsumura N., Baba T., Hamanishi J., Yamaguchi K., Murakami R., Taki M., Ukita M., Hosoe Y., Koshiyama M., Konishi I, & Mandai M. (2020). Anti-VEGF therapy resistance in ovarian cancer is caused by GM-CSF-induced myeloid-derived suppressor cell recruitment. British Journal of Cancer, 122(6), 778-788.

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Comprehensive Immune Cell Profiling

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Anti-human CD47 (clone B6H12, BD Biosciences), anti-mouse F4/80 (clone BM8, BioLegend), anti-Sirpα (clone P84, BioLegend), anti-mouse/human CD11b (clone M1/70, BioLegend), anti-mouse CD45 (clone 30-F11, BioLegend), anti-mouse MHC II (clone M5/114.15.2, BioLegend), anti-mouse CD206 (clone C068C2, BioLegend), anti-mouse CD80 (clone 16-10A1, BioLegend), anti-mouse CD86 (clone GL-1, BioLegend), anti-mouse PD-L1 (clone 10F.9G2, BioLegend), anti-mouse Gr1(clone RB6-8C5,BioLegend), anti-human CD14 (clone HCD14, BioLegend), and anti-human CD71 (clone CY1G4, BioLegend; clone OKT9, ThermoFisher; clone L01.1 and clone M-A712, BD Biosciences) were used for FACS analyses. Antibodies were Phycoerythrin (PE)-, PE/Cyanine7, APC, APC/Cyanine7, PerCP/Cyanine5.5, PE/Dazzle™ 594, Alexa Flour^® 700 or BV605 conjugated, or fluorophore-conjugated secondary antibodies were used. Annexin V (BD Biosciences), Sytox blue (ThermoFisher), 7-Aminoactinomycin D (7-AAD, ThermoFisher), or Zombie Violet™ Fixable Viability Kit (Biolegend) was used to exclude dead cells. Flow cytometry was performed using the BD LSRFortessa cell analyzers (BD).

Chen J., Cao X., Li B., Zhao Z., Chen S., Lai S.W., Muend S.A., Nossa G.K., Wang L., Guo W., Ye J., Lee P.P, & Feng M. (2021). Warburg Effect Is a Cancer Immune Evasion Mechanism Against Macrophage Immunosurveillance. Frontiers in Immunology, 11, 621757.

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Murine Immune Cell Profiling

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Murine samples of spleen tissues, lymph nodes and tumors were collected and prepared as previously described (23) . Cells were stained with the following antibodies: antimouse CD3 (clone 17A2, BioLegend, Cat#100221, RRID:AB_2057374), antimouse CD4 (clone GK1.5, Miltenyi Biotec, Cat#130-121-131, RRID:AB_2752219), antimouse CD8a (clone 53-6.7, BioLegend, Cat#100712, RRID:AB_312751), antimouse CD45 (clone 30-F11, BioLegend, Cat#103108, RRID:AB_312973, BD Biosciences, Cat#557659, RRID:AB_396774), antimouse F4/80 (clone BM8, BioLegend, Cat#123115, RRID:AB_893493), antimouse CD11b (clone M1/70, Miltenyi Biotec, Cat# 130-097-585, RRID:AB_2660136), antimouse Gr-1 (clone RB6-8C5, TONBO, San Diego, CA), antimouse Ly6C (clone HK1.4, BioLegend, Cat#128006, RRID:AB_1186135). Data was acquired using MACS Quant flow cytometer (Miltenyl Biotec, Bergisch Galdbach, Germany) and analyzed using FlowJo software (FlowJo, RRID:SCR_008520).

Mise Y., Hamanishi J., Daikoku T., Takamatsu S., Miyamoto T., Taki M., Yamanoi K., Yamaguchi K., Ukita M., Horikawa N., Abiko K., Murakami R., Furutake Y., Hosoe Y., Terakawa J., Kagabu M., Sugai T., Osakabe M., Fujiwara H., Matsumura N., Mandai M, & Baba T. (2022). Immunosuppressive tumor microenvironment in uterine serous carcinoma via CCL7 signal with myeloid-derived suppressor cells. Carcinogenesis, 43(7).

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