To examine the purity of lung macrophages and the population of M1/M2 cells after C. muridarum lung infection, freshly isolated lung mononuclear cells from NK-depleted and NK-intact mice were stained with anti-CD45-percp-cy5.5, anti-CD11b-FITC, anti-F4/80-PE, anti-iNOS-APC (M1) and anti-CD206-PE-cy7 (M2) antibodies (eBioscience). The raw data were collected using a BD FACSCanto 10C (BD Biosciences), and analyzed by Flowjo10.0 software.
Anti f4 80 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-F4/80-PE is a fluorescently-labeled antibody that binds to the F4/80 antigen, which is expressed on the surface of mature mouse macrophages. This antibody can be used for the identification and analysis of macrophage populations in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti gr1 fitc, by Thermo Fisher Scientific (4 mentions)
Anti cd11b fitc, by BD (4 mentions)
Anti ly6c apc cy7, by BioLegend (3 mentions)
Anti cd4 fitc, by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (3 mentions)
Anti cd11b apc, by Thermo Fisher Scientific (3 mentions)
Anti cd11b fitc, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (3 mentions)
Anti cd11b pe, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Anti cd45 apc cyanine7, by BioLegend (2 mentions)
Anti cd11c pe, by Thermo Fisher Scientific (2 mentions)
Anti gr 1 pe, by Thermo Fisher Scientific (2 mentions)
Anti cd45 efluor450, by Thermo Fisher Scientific (2 mentions)
7 aad, by BioLegend (2 mentions)
Anti cd3 a700, by Thermo Fisher Scientific (2 mentions)
Anti cd8 brilliantviolet605, by BioLegend (2 mentions)
Anti cd11b pe cy5, by Thermo Fisher Scientific (2 mentions)
Anti ly6g fitc, by BioLegend (2 mentions)
Anti cd64 apc, by BioLegend (2 mentions)
38 protocols using anti f4 80 pe
1
Lung Macrophage Purity and M1/M2 Profiling
2
Phagocytic Characterization of Peritoneal Cells
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Peritoneal cells were elicited from uninfected control or Eros−/− mice with 5 ml of PBS at 4°C. Cells were centrifuged at 1,200 rpm for 5 min in an Allegra X-15R centrifuge (Beckman Coulter). The pellet was red cell lysed by resuspension in lysis buffer containing 0.15 M ammonium chloride and 0.01 M sodium hydrogen carbonate. The cells were washed with PBS and resuspended in Optimem (Gibco) at 2 × 106 cells per well in a 24-well plate. S. Typhimurium SL1344 PssaG::GFP (which express GFP when Spi2 is active) were resuspended in PBS and incubated with 10% fresh mouse serum from control mice for 20 min at room temperature. They were added at 50 CFU per cell to the peritoneal cells and after 40 min, cells were harvested by gentle scraping, placed on ice, and incubated with anti–F480-PE (eBioscience) for 30 min at 4°C. The cells were washed in FACS buffer (PBS, 5% FBS, and 0.05% sodium azide) at 4°C and resuspended in PBS. FACS analysis was performed on a Cyan (Dako) or LSR-II (BD) flow cytometer. Peritoneal cells comprised 40–50% F480hi macrophages and ∼50–60% B-1 B cells in all strains studied. In some experiments, involving neutrophils a S. Typhimurium SL1344 PssaG::RFP strain was used.
3
Kidney Immune Cell Profiling
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Kidney single-cell suspensions were prepared via mechanical and enzymatic digestion as previously described (23 (link)). Suspensions were incubated with Fc Block anti-mouse CD16/32 (catalog no. 14-0161-85, eBioscience) for 15 min and then treated with anti-CD45-APC/Cyanine7 (catalog no. 103115, BioLegend), anti-CD11b-FITC (553310, BD), anti-F4/80-PE (12-4801-80, eBioscience), anti-CD206-Alexa Fluor 647 (565250, BD), and anti-CD86-eFluor 450 (48-0862-82; eBioscience) for 30 min at 4°C. Cells were washed with PBS, resuspended with 200 μl PBS, and detected using a Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). Data were analyzed using FlowJo software version 10.
4
Identification of Immune Cells in Tumor Microenvironment
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Immune cells were harvested from the tumor-draining lymph nodes of MC38-bearing mice and disrupted mechanically. The obtained cells were then passed through a 40 μm cell strainer to generate single-cell suspensions. The cells were incubated with anti-Gr-1-PE (12-5931, eBioscience), anti-F4/80-PE (12-3110, eBioscience), anti-CD11c-PE (12-0114, eBioscience), and anti-CD11b-FITC (11-0112, eBioscience) antibodies following the manufacturer's instructions. Subsequently, 20,000 events were recorded using BD (FACSCelesta), and the acquired data were analyzed using the FlowJo V10 software (Tree Star). The Gr-1+ cells were identified as neutrophils, whereas CD11b+ F4/80+ and CD11b+ CD11c+ cells were identified as macrophages and dendritic cells, respectively.
5
Urinary Immune Cell Profiling Protocol
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Bladders were digested at 37 °C for 30 min in RPMI-1640 with 10 mM HEPES, collagenase D, and DNAse (all from Sigma-Aldrich), forced through a 70 μm cell strainer (Corning), and washed with 5% fetal bovine serum (FBS, ThermoFisher Scientific, Durham, NC, USA) in PBS. Urines were diluted in fluorescence-activated single-cell sorting (FACS) Buffer (PBS supplemented with 5 mM EDTA and 3% FBS) for 30 min (pH neutralization), prior to staining. Single-cell suspensions resuspended on ice-cold FACS Buffer were stained with anti-CD45 eFluor450 (eBioscience, San Diego, CA, USA), anti-CD3-A700 (eBioscience), anti-CD4-PE/Cy7 (BioLegend, San Diego, CA, USA), anti-CD8-BrilliantViolet605 (BioLegend), anti-CD11b-PE/Cy5 (eBioscience), anti-Ly6C-APC/Cy7 (BioLegend), anti-Ly6G-FITC (BioLegend), anti-F4/80-PE (eBioscience), anti-CD64-APC (BioLegend), and 7-AAD (BioLegend). Purified anti-FcɣRIII/II (BioLegend) was used to block Fc-receptors. Data were acquired on LSR II flow cytometer (BD) and analyzed with FlowJo software v10.0 (BD).
6
Immunostaining of Macrophage Subsets
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Tissue samples were embedded in optimal cutting temperature compound (OCT) and snap frozen in liquid nitrogen. Tissue sections (4 μm) were fixed in ethanol/acetic acid fixative solution for 2–10 min, and then were stained with anti-F4/80-PE (eBioscience), CD206-PerCP-Cy5.5 (Biolegend) overnight at 4°C in a humidified chamber. After three times of washes, slides were stained with DAPI and mounted with Prolong Gold mounting reagent (Invitrogen). Confocal imaging was performed using Leica SP5 II confocal microscope.
7
Isolation and Immunophenotyping of Immune Cells
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Mesenteric and liver (celiac, portal) lymph nodes cells were isolated, washed, and counted. Cells were treated with FcR-block (Miltenyi Biotec) and stains were performed. Inflammatory monocytes: anti-CD11b-APC (Miltenyi Biotec, clone M1/70.15.11.5), anti-Ly6g-PacB (Biolegend, clone 18A), anti-F4/80-PE (eBioscience, clone BM8), anti-Ly6c-FITC (Novus Biologicals, clone HK1.4). T-cell hematopoiesis: anti-CD3e-APC (Miltenyi Biotec, clone 145-2C11), anti-TCRγδ-e450 (eBioscience, clone eBioGL3), anti-CD8a-PE (Miltenyi Biotec, clone 53-6.7), anti-CD4-FITC (Miltenyi Biotec, clone GK1.5). Dead cells were excluded via propidium iodide viability dye (Miltenyi Biotec). Data was acquired by the MACSQuant System (Miltenyi Biotec). Analyses were performed via FlowJo VX software (TreeStar).
8
Isolation of Kidney Macrophages in AAN Mouse Model
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Kidney macrophage isolation at day 14 of AAN was performed via kidney digestion and incubation with Fc Block (catalog No. 14-0161-85, eBioscience, San Diego, CA, USA). Cells were incubated with anti-CD45-APC/Cyanine7 (catalog no. 103115, BioLegend, San Diego, CA, USA), anti-CD11b-FITC (553310, BD, Franklin Lakes, NJ, USA), and anti-F4/80-PE (12-4801-80, eBioscience). CD45+ cells were first selected, and CD11b+F4/80+ cells were then gated for isolation via flow cytometry (BD FACSAria II). Total RNA was extracted from CD45+ CD11b+F4/80+ cells and reverse-transcribed via RT-qPCR analysis, as described below (23 (link)).
9
Single-Cell Isolation and Characterization
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To isolate single cells, tissue samples were minced and incubated in DMEM containing collagenase, type 4 (2 mg/ml, Worthington Biochemical, LS004188) and dispase II (1 mg/ml, SIGMA, D4693) for 100 min, with shaking at 37 °C. The digestion was stopped with DMEM containing 10% FBS and filtered through a 70-μm cell filter (BD). The cells were then diluted 1:100 with Zombie Aqua™ dye in PBS, resuspended, and incubated for 15 min at RT in the dark. The single-cell suspension was incubated with Fc Block for 15 min at 4 °C. After washing, a mixture of cell surface antibodies was added, followed by incubation for an additional 30 min at 4 °C in the dark. We used the following antibodies: anti-CD45-FITC (eBioscience, California USA), anti-CD3-PerCP-Cy5.5 (eBioscience, California, USA), anti-CD11b-Pacific Blue (eBioscience, California, USA), anti-CD11c-PE-Cy7 (eBioscience, California, USA), anti-F4/80-PE (eBioscience, California, USA), and anti-ly6G-PerCP-Cy5.5 (eBioscience, California, USA). Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions and detected using flow cytometry (FACSCalibur, BD, San Jose, CA).
10
Murine Myeloid-Derived Suppressor Cell Characterization
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RPMI 1640, DMEM, FBS, and antibiotics were obtained from Life Technologies. Recombinant murine GM-CSF, anti–TGFB1-APC, and IL-2 were obtained from R&D Systems. The following antibodies were purchased from eBioscience: anti-Gr1 FITC, anti-Gr1 PE, anti-F4/80+ PE, anti-Cd11b+ PE, anti-CD11c+ APC, anti–PD-L1 PE, anti–PD-L1 FITC, anti–PD-L1 APC, anti–PD-L2 PE, anti-CD80 FITC, anti-CD86 FITC, anti–PD-1 APC, anti–CTLA-4 APC, anti-CD4 FITC, anti-CD8 FITC, anti–IFN-γ PE-cy7, anti-IL6 FITC, anti-IL10 APC, anti-IL12p70 PE, and functional grade anti–PD-L1 (MIH5) neutralizing antibody. For blocking, control antibody (IgG; rat IgG2b K Isotype Control Functional Grade Purified; eBioscience), anti–mouse IL-6 Functional Grade Purified neutralizing antibody (eBioscience), or anti–mouse IL-10 Functional Grade Purified neutralizing antibody (eBioscience) were used.
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