Immunofluorescence (IF) staining was performed to identify the infiltrated macrophages in the mouse myocardium by incubating the tissues with mouse anti-F4/80 (1:100, Biolegend, CA, USA) at 4 °C overnight. The nuclei of the cells were stained with a mounting medium (antifading, with DAPI, Solarbio, Beijing, China). Finally, the cells were observed and photographed with a Leica DM6 B Orth fluorescence microscope.
Anti mouse f4 80
Manufactured by BioLegend
Sourced in United States
Anti-mouse F4/80 is a mouse monoclonal antibody that recognizes the F4/80 antigen, a glycoprotein expressed on the surface of mouse macrophages. The antibody can be used for the identification and isolation of mouse macrophages in flow cytometry and immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
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Anti mouse cd3, by BioLegend (2 mentions)
Anti mouse cd4, by BioLegend (2 mentions)
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Anti mouse ly6c, by BD (1 mentions)
Anti human cd33, by BD (1 mentions)
25 protocols using anti mouse f4 80
1
Macrophage Identification in Mouse Myocardium
2
Immunohistochemical Staining of Skin Tissues
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IHC staining in skin tissues were performed as previously described (18 (link)). Briefly, tissue sections were stained with rabbit anti-MPO (1:200, Thermo), mouse anti-F4/80 (1:200, BioLegend), rabbit anti-IL-6 (1:600, Proteintech), and mouse anti-p-STAT3 (1:200, CST). Then, DAB chromogenic system was used for final chromogen. Images of areas of interest were collected by Olympus IX73-A21PH microscope (Olympus, Japan). Epidermal p-STAT3 positive cells were quantitified as the number of positive cells per mm migrating epidermis. Quantifications for MPO, F4/80, IL-6, and stromal p-STAT3 positive cells were performed by using Image J by calculating 6-8 40× high power field photos per mouse.
3
Intestinal Tumor Polyp Analysis
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The 14-week-old ApcMin/+ mice were immunized with VLP-hCEA via oral gavage. After 48 h, intestinal tumour polyps from small intestine were collected and snap frozen. Frozen sections were then subjected for co-staining with 4,6-diamidino-2-phenylindole (DAPI), FAM-FLICA caspase-1 probe and A594-conjugated anti-mouse F4/80 (Biolegend) or DAPI, FAM-FLICA caspase-1 probe and anti-mouse EpCAM (Abcam) antibody. A594-conjugated anti-Rat IgG was used as the secondary antibody.
4
Flow Cytometry Protocol for Cell Characterization
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The detailed flow cytometry procedure was performed as previously described [31 (link)]. The following antibodies were used: anti-mouse IFNγ (BD Biosciences), anti-mouse CD45 (BD Biosciences), anti-mouse Gr-1 (TONBO Biosciences), anti-mouse Ly6G (TONBO Biosciences), anti-mouse ly6C (BD Biosciences), anti-mouse CD11b (TONBO Biosciences), anti-mouse F4/80 (BioLegend), anti-human CD11b (TONBO Biosciences), anti-human CD33 (BD Biosciences), and anti-human HLA-DR (BD Biosciences). Detailed information on the antibodies is provided in Additional file 2 : Table S2. Briefly, the cells were digested and suspended as single cells, washed with PBS, and then resuspended in cell stabilizing buffer (BioLegend cat. No. 420201); the supernatant was discarded, and the sample was centrifuged at 350 g for 5 min. Then, 5 μl of Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301) and the antibodies were added and cultured for 15-20 min at room temperature. Finally, the cells were assessed by flow cytometry. For intracellular staining of IFNγ, fixation buffer (BioLegend Cat. No. 420801) was added and incubated for 20 min at room temperature. Then, the cells were washed with Intracellular Staining Perm Wash Buffer (BioLegend Cat. No. 421002), resuspended and centrifuged. IFNγ antibody was added, and the cells were cultured for 20 min and finally assessed by flow cytometry.
5
Phagocytosis of Raji Cell Fragments by RAW264.7 Macrophages
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RAW264.7 cells (Procell,Wuhan,China) were cultured in DMEM (Gibco, Australia) with 10% FBS (Gibco, Australia) without penicillin‒streptomycin. Raji cells were maintained in RPMI-1640 medium (BI, Israel) containing 10% FBS. Raji cells (Procell,Wuhan,China) were prepared at a density of 2X106 cells/mL. A total of 500 µL of the cell suspension and 500 µL of 5 µM CFSE (Biolegend) working solution were mixed. After being incubated for 10 min, 5 mL of complete medium was added to halt the staining, followed by two washes with cold PBS. The cells were resuspended in complete medium and placed in the incubator for 30 min to allow the cells to release excess CFSE. Raji cells were resuspended in 150 μL of PBS and lysed by freezing and thawing. RAW264.7 cells were plated into the lower wells of Transwell chambers and cocultured with CD146±UCMSCs in the upper wells. Raji-CFSE fragments were added to the lower chamber. RAW264.7 cells were digested for 30 min, 1 h and 2 h, and the supernatant was collected. The anti-mouse F4/80 (Biolegend) antibody was used for labelling. The percentage of F4/80+CFSE+ cells in total macrophages was measured for phagocytosis analysis.
6
Immune Cell Phenotyping Protocol
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Live/dead cells were discriminated using with eFluor 780 fixable viability dye (eBioscience,) for 30 mins at room temperature covered from light. Cells were then blocked with anti-CD16/CD32 (eBioscience) for 20 min. Cells were subsequently stained for extracellular markers anti-mouse CD11b (30-H12), anti-mouse CD45 (30-F11), anti-mouse Ly6G (1A8), anti-mouse CD3 (145-2C11), anti-mouse CD4 (L3T4), anti-mouse CD11c (N418) anti-mouse F4/80 (BM8), anti-mouse lineage cocktail (Biolegend, 133302), anti-mouse Sca-1 (D7), anti-mouse ST1 (D1H9), anti-mouse CD127 (A7R34), anti-mouse 90.2 (30-H12), anti-mouse CD25 (3C7), anti-mouse Siglec-F (S17007L), anti-mouse CD8α (25–0081) for 30 minutes. Samples were fixed for 15 min with 2% PFA. Sample acquisition was conducted using BD LSRFortessa and analysed using FlowJo Software. The IL-4R neutralizing antibody (kindly provided from of Dr. Manel Jordana, McMaster University) was labelled with NHS-Fluorescein (Thermo Scientific).
7
Antibody Profiling for Tissue Analysis
8
Flow Cytometric Analysis of Liver Immune Cells
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Mice were sacrificed and the liver was harvested for analysis ex vivo as previously described (Park et al., 2017 (link)). Cells were isolated and stained with anti-mouse CD45 (1:100 dilution, Biolegend, #103116), anti-mouse F4/80 (1:100 dilution, Biolegend, #123108), anti-mouse CD206 (1:100 dilution, Biolegend, #141720), and anti-mouse CD11c (1:100 dilution, Biolegend, #117310), and then tested using flow cytometry (LSRFortessaTM cell analyzer, BD, United States). Data were analyzed with FlowJo V10.6.
9
Immune Cell Profiling in Tooth Extraction
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The tissue around the tooth sockets and peripheral blood were collected during the inflammatory phase of tissue 1 week after tooth extraction. Peripheral blood was processed by lysing the red blood cells. The tissue was digestion, centrifugation and filtration. Finally, the single cell suspension was stained for immune cell analysis. The cells were incubated with purified anti‐mouse CD45, anti‐mouse F4/80, anti‐mouse CD11b, anti‐mouse CD11c, anti‐mouse Ly6C, anti‐mouse Ly6G and anti‐mouse CD3 (Biolegend) at 4 °C for 30 min. Stained cells were tested on FACS Symphony (BD Biosciences), and data were analysed with FlowJo software.
10
Murine BMDM Characterization by FACS
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Following polarization of BMDMs, ~1–2 × 106 cells were subjected to FACS surface and intracellular staining protocol. Briefly, cells were initially suspended in FACS buffer solution (0.3% BSA in PBS; McMaster University, Hamilton, ON, Canada). Non‐antigen‐specific binding of immunoglobulins to Fcγ II/III receptor on BMDMs was blocked using purified rat anti‐mouse CD16 (Mouse FC Block) (BD Pharmingen, Mississauga, ON, Canada; Cat#553142). Next, cells were subjected to surface antibody staining solution, containing a mix of anti‐mouse F4/80 and anti‐mouse CD206 (MMR) (BioLegend, San Diego, CA, USA; Cat#123133 and 141723). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences, Mississauga, ON, Canada; Cat#554722) prior to performing intracellular staining with anti‐mouse/human arginase‐1 (1:5) (R&D Systems, Toronto, ON, Canada; Cat#IC5868A). Intracellular staining antibody solution was made up in 1× BD Perm/Wash buffer solution (BD Biosciences, Cat#554723). Cells were finally resuspended in FACS buffer for FACS and data were collected using BD LSRFortessa and FACSDiva software from BD Biosciences. Data were analyzed using FlowJo Software from Treestar.
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