RAW264.7 cells in the logarithmic growth phase were spread to a 6-well plate at 106 cells/well. After at least 6 h, the cells were completely attached to the wall, and the lysate and control were added separately. After 24 h of treatment, RAW264.7 cells were stained with FITC anti-mouse F4/80 (clone: FJK-16s, Invitrogen, Waltham, Massachusetts), APC anti-mouse CD86 (clone: GL-1, Biolegend, San Diego, CA) and PE anti-mouse CD206 (clone: MR6F3, Invitrogen, Waltham, Massachusetts) according to the protocol of the antibodies (M1 macrophage: F4/80+CD86+, M2 macrophage: F4/80+CD206+) and subjected to flow cytometry (LSR II, BD). Detection of SIRPα on macrophages in spleen from mouse used FITC anti-mouse F4/80, APC anti-mouse CD11b (clone: M1/70, Biolegend, San Diego, CA), and PE anti-mouse SIRPα (clone: P84, Biolegend, San Diego, CA). All flow cytometry used to detect macrophage typing in this study was set up with an antibody isotype control group (PE Rat IgG2a, clone: RTK2758, Biolegend, USA; APC Rat IgG2a, clone: RTK2758, Biolegend, USA; FITC Rat IgG2a, clone: RTK2758, Biolegend, USA).
Anti mouse f4 80 fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse F4/80-FITC is a fluorescently-labeled antibody that binds to the F4/80 antigen, a member of the EGF-TM7 family of cell surface receptors. It is commonly used as a marker for the identification and isolation of mouse macrophages in flow cytometry and immunofluorescence applications.
Automatically generated - may contain errors
Lab products found in correlation
Pe anti mouse cd8, by Thermo Fisher Scientific (2 mentions)
Apc anti mouse cd206, by BioLegend (2 mentions)
Pe anti mouse cd86, by BD (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Guava easycyte 12ht benchtop flow cytometer, by Merck Group (2 mentions)
Eflour 450 anti mouse cd11b, by Thermo Fisher Scientific (2 mentions)
Mouse trustain fcx, by BioLegend (2 mentions)
Anti mouse cd11b apc, by Thermo Fisher Scientific (2 mentions)
Anti mouse ly6g fitc, by Thermo Fisher Scientific (2 mentions)
Anti mouse ly6c pe, by Thermo Fisher Scientific (2 mentions)
Fitc anti mouse f4 80, by BioLegend (1 mentions)
Apc anti mouse cd11b, by BioLegend (1 mentions)
Apc rat igg2a, by BioLegend (1 mentions)
Apc anti mouse cd86, by BioLegend (1 mentions)
Pe rat igg2a, by BioLegend (1 mentions)
Fitc rat igg2a, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
11 protocols using anti mouse f4 80 fitc
1
Macrophage Polarization Analysis by Flow Cytometry
2
Tumor Immune Cell Profiling
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Tumors were collected at 15 days after s.c. injection of B16F10 cells. The tumor masses were minced into 3–5 mm2 slices and dissociated by incubating in 1 mg/ml collagenase type IV (Sigma, C5138) and 20 mg/ml DNase (Sigma, D5025) for 30 min with rotation. The resulting mixture was suspended in FACS buffer (5% FBS in PBS) and passed through a 70-μm nylon cell strainer (SPL, 93070) to obtain a single-cell suspension. Fc receptors were blocked using TruStain FcX (Biolegend, 101319) according to the manufacturer’s protocol, and cells were stained with the following fluor-conjugated antibodies for 1 h on ice: PE anti-mouse CD8 (Invitrogen, 12-0081-82), PerCP anti-mouse CD3 (Invitrogen, 45-0031-82), PE anti-mouse NK1.1 (Invitrogen, 12-5941-82), PE anti-mouse CD11c (Invitrogen, 12-0114-82), FITC anti-mouse F4/80 (Invitrogen, 11-4801-82), and PE anti-CD206 antibodies (Biolegend, 141706). To detect Intracellular IFNγ, cells were fixed with 4% paraformaldehyde (Sigma, F8775) for 15 min and were permeabilized with 0.2% Triton X-100 (Sigma, T9284) for 10 min and were probed with FITC anti-mouse IFNγ (Biolegend, 505805). Data were acquired on a FACSCantoII (BD Biosciences) and were analyzed using FACSDiva software.
3
Flow Cytometry Characterization of Macrophage Subsets
4
Characterization of Tumor-Associated Macrophages
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Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in Hank's Balanced Salt Solution (HBSS) (Gibco) for 15 min at RT, then blocked in 100 μL of FACS Buffer (HBSS without calcium, 2% FBS and 1mM EDTA) with 1% bovine serum albumin (BSA) (Sigma) for 20 min at 4°C. Cells were washed with FACS buffer between each step. Cells suspensions of 50 μL were incubated for 15 min at RT with 0.5 μL Mouse TruStain FcX (Biolegend, 101319) to prevent nonspecific Fc receptor binding. Samples were immediately stained with the following primary antibodies for 30 min at 4°C: 0.125μg/100μL eFlour 450 anti-mouse CD11b (Thermo Fisher Scientific, Clone M1/70), 0.5μg/100μL FITC anti-mouse F4/80 (Thermo Fisher Scientific, Clone BM8), 0.5μg/100μL PE anti-mouse CD86 (BD Biosciences Clone GL1), and 0.5μg/100μL APC anti-mouse CD206 (BioLegend, Clone C068C2). Cells were washed 2x with FACS buffer and analyzed using a Guava EasyCyte 12HT benchtop flow cytometer (MilliporeSigma). Flow cytometry plots were analyzed using FlowJo v10.7.1 software. Macrophages were characterized as CD11b+ F4/80+ populations. Within the gated macrophage population, M1/M2 gates were made using a control sample for each tumor, stained only for CD11b and F4/80 in the absence of M1/M2 markers to account for background fluorescence. M1 macrophages were characterized as CD86+ while M2 macrophages were CD206+.
5
Surface Cell Staining Protocol
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For surface cell staining, 5 × 105 cells were incubated for 15–20 min with anti-mouse CD16/CD32 to block non-specific binding of immunoglobulins to Fc receptors expressed on monocytes, macrophages, and granulocytes (Biolegend, clone 93). Labeling was performed for 30 min with specific antibodies. The following antibodies were used: PE anti-mouse CD11b (Biolegend, clone M1/70), FITC anti-mouse F4/80 (eBioscience, clone BM8), and FITC anti-mouse Ly6G (Biolegend, clone 1A8). All staining was done on ice in PBS supplemented with 2% BSA followed by washing once in cold PBS. The FACS Vantage system (Becton-Dickinson, San Jose, CA, USA) was used to analyze samples.
6
Multicolor Flow Cytometry for Immune Cell Profiling
7
Immunohistochemical Analysis of Colonic Macrophages and Neutrophils
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Paraffin-embedded colon samples were sectioned. After dewaxing and hydration, antigen retrieval was achieved by immersing the sections in a citrate buffer. Slides were then blocked with avidin/biotin agent (Vector Laboratories, Burlingame, CA, USA). To analyze the infiltration of macrophages and neutrophils, colonic slides were stained with FITC anti-mouse F4/80 (a murine macrophage-restricted cell surface glycoprotein) (eBioscience, San Diego, CA, USA) and PE/Cyanine5-labeled anti-mouse Ly6G (lymphocyte antigen 6 complex locus G) antibodies (Biolegend, San Diego, CA, USA). The nuclei were counterstained with DAPI (Vector Laboratories). Slices were sealed with anti-fluorescence quenching solution and analyzed under a fluorescence microscope (Leica DM3000; Wetzlar, Germany). The mean number of F4/80+ cells detected in each high-power field was calculated by counting four fields from each sample (samples from three mice per group were counted).
8
Murine Myeloid-Derived Suppressor Cell Analysis
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Bone marrow (BM) cells were flushed from mouse femurs and tibiae and prepared as single-cell suspension after lysing the red blood cells. Peripheral blood mononuclear cells (PBMC) in mouse blood samples were isolated with Ficoll-Hypaque (Axis-Shield). All cells labeled with antibodies were immediately analyzed on a FACS Calibur (BD Biosciences, Mountain View, CA, USA).
For the analysis of MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, and anti-mouse Gr1-PE.
For the analysis of PMN-MDSCs and M-MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Ly6G-FITC, and anti-mouse Ly6C-PE.
For the analysis of immature or undifferentiated markers CD11c, F4/80, CD80, and MHCII on MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Gr1-PE or anti-mouse Gr1-FITC, and anti-mouse CD11c-FITC, anti-mouse F4/80-FITC, anti-mouse CD80-PE, or anti-mouse MHCII-PE.
For the analysis of MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, and anti-mouse Gr1-PE.
For the analysis of PMN-MDSCs and M-MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Ly6G-FITC, and anti-mouse Ly6C-PE.
For the analysis of immature or undifferentiated markers CD11c, F4/80, CD80, and MHCII on MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Gr1-PE or anti-mouse Gr1-FITC, and anti-mouse CD11c-FITC, anti-mouse F4/80-FITC, anti-mouse CD80-PE, or anti-mouse MHCII-PE.
9
Investigating Cellular Mechanisms with Curcumin
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Curcumin, piperine, actin antibody and protein G magnetic beads were purchased from Sigma-Aldrich, Inc (St. Louis, MO), Dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Molecular Probes (Eugene, OR). The following antibodies were purchased from eBiosciences (San Diego, CA): anti-mouse-Ly6G and Ly6C PE, anti-mouse-CD11b APC, anti-mouse F4/80 FITC, anti-mouse CCR7 APC, anti-mouse Ly6G FITC, anti-mouse CD4 FITC, anti-mouse CD8 PE, anti-mouse Ly6C PE, isotype control antibodies. Annexin V Apoptosis kit, Bax 6A7 antibody, anti-mouse-IFN-γ APC were obtained from BD Biosciences (San Jose, CA). Biotinylated anti-Gr1, anti-Ly6C, anti-Ly6G and streptavidin microbeads were purchased from Miltenyi Biotech (Auburn, CA). Anti-mouse Dectin-1 was purchased from AbD Serotec. Clusterin antibodies were purchased from Upstate (Millipore, Billerica, MA). Clusterin siRNA and Scrambled negative control were purchased from OriGene (Rockville, MD). MitoTracker Red CMXRos, Alexa 488 anti-mouse secondary antibody and Lipofectamine 2000 were purchased from Invitrogen (Grand Island, NY).
10
Lung Cell Population Characterization
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The adipose tissues and bronchus of each group of lungs were removed. The lungs were then cut into pieces and digested in RPMI 1640 medium (VivaCell, Shanghai, China) containing 10% FBS (Sperikon Life Science & Biotechnology Co., Ltd., Chengdu, China) and collagenase VIII (250 U/mL; Sigma Aldrich, Saint Louis, MO, USA) at 37 °C for 30 min. Cell surface staining was performed by incubating the cells with the indicated antibodies on ice for 15 min. Cells were washed and analyzed on an LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) using FlowJo software (BD Biosciences, San Diego, CA, USA).
To measure the percentages of CD4+ and CD8+ cells among CD3+ mononuclear cells, the cells were incubated with anti-mouse CD3e-FITC (1:1200, eBioscience); anti-mouse CD4-APC (1:400, eBioscience); anti-mouse CD8a-PE (1:1600, BioLegend); anti-mouse CD45R-PerCP-Cyanine5.5 (1:800, eBioscience) for 20 min at room temperature.
To measure the percentages of macrophages and dendritic cells, cells were incubated with anti-mouse I-A/I-E-PE/Cyanine7 (1:3200, BioLegend); anti-mouse F4/80-FITC (1:100, eBioscience); anti-mouse CD11b-eFluor™ 450 (1:800, eBioscience); anti-mouse CD11c-PerCP-Cyanine5.5 (1:200, eBioscience); anti-mouse Ly-6C-APC (1:800, eBioscience) for 20 min at room temperature.
To measure the percentages of CD4+ and CD8+ cells among CD3+ mononuclear cells, the cells were incubated with anti-mouse CD3e-FITC (1:1200, eBioscience); anti-mouse CD4-APC (1:400, eBioscience); anti-mouse CD8a-PE (1:1600, BioLegend); anti-mouse CD45R-PerCP-Cyanine5.5 (1:800, eBioscience) for 20 min at room temperature.
To measure the percentages of macrophages and dendritic cells, cells were incubated with anti-mouse I-A/I-E-PE/Cyanine7 (1:3200, BioLegend); anti-mouse F4/80-FITC (1:100, eBioscience); anti-mouse CD11b-eFluor™ 450 (1:800, eBioscience); anti-mouse CD11c-PerCP-Cyanine5.5 (1:200, eBioscience); anti-mouse Ly-6C-APC (1:800, eBioscience) for 20 min at room temperature.
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