F4 80 fitc clone bm8

Manufactured by BioLegend

F4/80-FITC (clone BM8) is a fluorescently-labeled monoclonal antibody that recognizes the F4/80 antigen. F4/80 is a cell surface glycoprotein expressed on mature mouse macrophages. This product can be used for the identification and enumeration of mouse macrophages by flow cytometry.

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F4/80 (349 citations) F4/80-FITC (57 citations) F4/80 (BM8, (56 citations) F4/80 (clone BM8, (35 citations) Clone BM8 (20 citations) F4/80-FITC (BM8, (2 citations)

6 protocols using f4 80 fitc clone bm8

Multiparametric Immune Cell Analysis

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Cells were stained using the following fluorophore conjugated anti-mouse antibodies: WGA-488 (Invitrogen), Ly6G-FitC (clone 1A8, BioLegend), Ly6G-BV510 (clone 1A8, BioLegend), Ly6G-PE (clone 1A8, BioLegend), CD11c-BV605 (clone N418, BioLegend), F4/80-FitC (clone BM8, BioLegend), F4/80-PE (clone BM8, BioLegend), CD11b-PE (clone M1/70, BioLegend), Ly6C-PE Cy7 (clone HK1.4, BioLegend), CD63-APC (clone NVG-2, BioLegend), CD9-APC (clone MZ3, BioLegend), IL-1α-PE (clone ALF-161, BioLegend), and IL-1β-APC (clone NJTEN3, ThermoFisher Scientific). Antibodies were diluted in wash buffer (PBS with 1% BSA and 2 mM EDTA). Cells were stained for 20 minutes at 4°C, washed with wash buffer, fixed for 15 minutes in BD Biosciences cytofix/cytoperm, and permeabilization prior to intracellular staining. ACEA Novocyte was used for flow cytometry, and Novoexpress software was used for subsequent analysis. AMNIS ImageStream was used for imaging flow cytometry and the AMNIS IDEAS software was used to calculate the colocalization coefficient.

Ratitong B., Marshall M, & Pearlman E. (2021). β-Glucan-stimulated neutrophil secretion of IL-1α is independent of GSDMD and mediated through extracellular vesicles. Cell reports, 35(7), 109139.

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Neonatal Lung Immune Cell Profiling

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Neonatal lung single‐cell suspensions were used for all immunostaining. Panels of monoclonal antibodies (purchased from BD Bioscience unless otherwise stated) were developed to enable phenotypic characterisation of leucocytes of myeloid: CD45‐PerCP (clone 30‐F11), CD11b‐v500 (clone M1/70), CD11c‐AF700 (clone HL3), CD19‐BV786 (clone 1D3), CD103‐PE (clone M290), CD301‐PE‐Cy7 (clone MGL1/MGL2; BioLegend), F4/80‐FITC (clone BM8; BioLegend, San Diego), Ly6G/C‐APC‐Cy7 (clone RB6‐8C5), I‐A/I‐E‐AF647 (clone M5/114.14.2) and B220/CD45R‐PE‐CF594 (clone RA3‐6B2) and lymphoid: CD45‐PerCP (clone 30‐F11), NKp46‐PE‐Cy7 (clone 29A1.4; BioLegend), CD19‐BV786 (clone 1D3), CD3‐FITC (clone 17A2), CD4‐v500 (clone RM4‐5), CD8α‐BV650 (clone 53‐6.7), CD25‐APC‐Cy7 (clone PC61) and Foxp3‐PE (clone FJK‐16s) lineages. Intracellular staining for Foxp3 was performed using an intracellular Foxp3/Transcription Factor Staining Buffer Kit (eBioscience, San Diego). All samples were kept as individuals. Immune cell phenotypic characterisation was performed using the FlowJo software (version 10.6.1; BD Bioscience). Fluorescent minus one staining controls were used for all panels where necessary. Flow cytometry data quality was based on primary time gates to ensure appropriate laser delay (pre‐determined by automated CS&T) during sample acquisition.

Lauzon‐Joset J., Mincham K.T., Scott N.M., Khandan Y., Stumbles P.A., Holt P.G, & Strickland D.H. (2021). Protection against neonatal respiratory viral infection via maternal treatment during pregnancy with the benign immune training agent OM‐85. Clinical & Translational Immunology, 10(7), e1303.

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Multiparametric Immune Cell Analysis

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Ratitong B., Marshall M, & Pearlman E. (2021). β-Glucan-stimulated neutrophil secretion of IL-1α is independent of GSDMD and mediated through extracellular vesicles. Cell reports, 35(7), 109139.

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CFSE Labeling and Macrophage Interaction

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Lewis cancer cells were incubated with 5 nM carboxyfluorescein succinimidyl ester (CFSE) at 37 °C in the dark for 20 min. After washing away free CFSE, the CFSE-labeled Lewis cells were collected, and the cell concentration was adjusted. The collected cells were added to bone marrow-derived macrophages (BMDMs) at a tumor cell:macrophage ratio of 1:4 and cultured for 2 h. The cells were then incubated with F4/80-FITC (clone BM8, BioLegend) for flow cytometry analysis.

Sheng Y., Zhang B., Xing B., Liu Z., Chang Y., Wu G, & Zhao Y. (2023). Cancer-Associated Fibroblasts Exposed to High-Dose Ionizing Radiation Promote M2 Polarization of Macrophages, Which Induce Radiosensitivity in Cervical Cancer. Cancers, 15(5), 1620.

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FACS Analysis of Accessory Molecules on RAW264.7 Cells

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The detection of accessory molecule expression on RAW264.7 cells by FACS analysis was performed as follows. Briefly, RAW264.7 cells were harvested by accutase or EDTA dissociation buffer and washed with FACS buffer (3% bovine serum albumin (BSA) and 0.1% sodium azide in DPBS). The cells were stained with the optimal concentrations of fluorochrome-conjugated antibodies for 30 min. The antibodies used in these experiments were Fas ligand-APC (eBioscience, San Diego, CA), CD95-FITC (BD Pharmingen, San Diego, CA), and F4/80-FITC (clone: BM8; BioLegend, San Diego, CA). One hundred thousand cells per treatment were acquired and analyzed using a BD FACSVerse flow cytometer and FlowJo software (version 10.7, BD Ashland, OR). Cells were gated using forward scatter vs. sideward scatter properties to exclude debris and doublets. The mean fluorescence intensity (MFI) of the whole macrophage population was analyzed.

Lai T.Y., Cao J., Ou-Yang P., Tsai C.Y., Lin C.W., Chen C.C., Tsai M.K, & Lee C.Y. (2022). Different methods of detaching adherent cells and their effects on the cell surface expression of Fas receptor and Fas ligand. Scientific Reports, 12, 5713.

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Murine Immune Cell Isolation and Analysis

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DMEM, RPMI-1640, fetal bovine serum (FBS), penicillin-streptomycin (PS), Trypsin-EDTA, and phosphate-buffered saline (PBS) were purchased from Gibco. Murine GM-CSF and IL-6 from PeproTech, artemisinin (HY-B0094) from MedChemExpress, InVivoMab anti-mouse PD-L1 antibody (clone 10F.9G2) from BioXCell, dimethyl sulfoxide (DMSO, D2650) and concanavalin A (Con A, C2272) from Sigma-Aldrich, and carboxyfluorescein succinimidyl ester (CFSE) from Invitrogen were obtained. The following are fluorescein-conjugated anti-mouse antibodies: Gr-1-PerCP-Cy5.5 (clone RB6-8C5), Gr-1-FITC (clone RB6-8C5), CD11b-APC (clone M1/70), CD11b-PE (clone M1/70), CD45-Brilliant Violet 510 (clone 30-F11), Ly6C-PE-Cy7 (clone HK1.4), Ly6G-APC-Cy7 (clone 1A8), CD11c-Brilliant Violet 421 (clone N418), F4/80-FITC (clone BM8), CD3-APC (clone 17A2), CD3-FITC (clone 17A2), CD4-APC-Cy7 (clone GK1.5), CD4-APC (clone GK1.5), CD8a-PE (clone 53-6.7), CD8a-Brilliant Violet 605 (clone 53-6.7), CD19-Alexa Fluor 700 (clone 6D5), NK1.1-PE-Cy7 (clone PK136), CD25-APC (clone 3C7), DR5-PE (clone MD5-1), Annexin V-FITC, and propidium iodide (PI) solution were from Biolegend. LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit was from Invitrogen, and Foxp3-PE (clone R16-715) was from BD Pharmingen.

Zhang M., Wang L., Liu W., Wang T., De Sanctis F., Zhu L., Zhang G., Cheng J., Cao Q., Zhou J., Tagliabue A., Bronte V., Yan D., Wan X, & Yu G. (2022). Targeting Inhibition of Accumulation and Function of Myeloid-Derived Suppressor Cells by Artemisinin via PI3K/AKT, mTOR, and MAPK Pathways Enhances Anti-PD-L1 Immunotherapy in Melanoma and Liver Tumors. Journal of Immunology Research, 2022, 2253436.

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