Cells were labeled using the anti-mouse antibodies: CD11b-PE (concentration 1:400), iNOS-Alexa Fluor 488 (concentration 1:100), and CD206-APC (concentration 1:100) (Biolegend). We collected 10,000 cell events per sample, and we performed unstained control as well as isotype controls. Labeled cells were examined on the LSR II Analyzer (BD) in the Stanford Shared FACS Facility, and data was analyzed with FlowJo software (Tree Star, Ashland, OR). Dead cells were excluded by ethidium monoazide (EMA) staining, and appropriate isotype controls were used. Quadrants were drawn using the M0 isotope control.
Cd206 apc
Manufactured by BioLegend
Sourced in United States
CD206-APC is a flow cytometry reagent that detects the expression of the CD206 antigen, also known as the macrophage mannose receptor. CD206 is a type I transmembrane glycoprotein that is primarily expressed on the surface of macrophages and dendritic cells. This reagent is conjugated with the fluorescent dye Allophycocyanin (APC), which can be detected using flow cytometry.
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Lab products found in correlation
Cd86 pe, by BioLegend (10 mentions)
Cd11b fitc, by BioLegend (8 mentions)
F4 80 pe, by BioLegend (7 mentions)
Lsr 2 flow cytometer, by BD (6 mentions)
Cd11b pe cy7, by BioLegend (6 mentions)
Gr 1 pe, by BioLegend (6 mentions)
Cd11c apc cy7, by BioLegend (5 mentions)
F4 80 af488, by BioLegend (5 mentions)
Cd45 pe, by Thermo Fisher Scientific (4 mentions)
Cd11b fitc, by BD (4 mentions)
F4 80 pe cy7, by Thermo Fisher Scientific (4 mentions)
F4 80 fitc, by BioLegend (4 mentions)
Cd45 fitc, by Thermo Fisher Scientific (4 mentions)
Cd86 fitc, by BioLegend (4 mentions)
Cd11b pe, by BioLegend (3 mentions)
Cd4 fitc, by Thermo Fisher Scientific (3 mentions)
Collagenase, by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (3 mentions)
Cd14 apc cy7, by BD (3 mentions)
Cd80 pe, by BioLegend (3 mentions)
64 protocols using cd206 apc
1
Multicolor Flow Cytometry Analysis
2
Flow Cytometric Analysis of Microglia in Brain Tissue
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First, brain cortex was isolated from experimental mice on the ice. Secondly, cell suspension was prepared via gridding brain tissue through 70 μm filters. Then sing cells were centrifuged at 3000 g for 10 minutes at 4°C. After centrifugation, we removed the supernatants and washed the cells with 100 μl PBS, followed by centrifugation for 10 minutes at 1000 g. Cells were suspended by 100 μl PBS again, and incubated with primary antibodies of CX3CR1-PE (1:200, Biolegend, San Diego, CA), CD16/32-FITC (1:100, Ebioscience, Franklin Lakes, NJ), and CD206-APC (1:50, Biolegend) at room temperature for 15 minutes. These cells were then washed with 3 ml PBS 0.1% sodium azide and 5% fetal bovine serum, followed by centrifugation for 5 minutes at 300 g suspend the cells in PBS and analyzed immediately using a Flow Activated Cell Sorter (FACS; BD Bioscience).
3
Comprehensive Lung Cell Immunophenotyping
4
Tumor Macrophage Profiling via Flow Cytometry
5
Phenotypic Characterization of Macrophages and Mesenchymal Stem Cells
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After culturing with the supernatant from each group of stem cells for 24 h, the macrophages were collected and the density was adjusted to 1 × 106/ml, after which the following monoclonal fluorescent antibodies were added: CD206-APC (Rat IgG, Monoclonal, BioLegend Cat. No. 141708), CD86-PE (Rat IgG, Monoclonal, BioLegend Cat. No. 105008), Arg-1-PE (Syrian Hamster IgG, Monoclonal, BioLegend Cat. No. 138408) and iNOS-FITC (Rat IgG, Monoclonal, BioLegend Cat. No. 696802). For identification of the MSCs, a suspension of P3 generation UC-MSCs was collected, after the following monoclonal fluorescent antibodies were added: CD31-ECD, CD34-ECD, CD45-ECD, HLA-DR-ECD, CD29-ECD, CD90-ECD, CD73-ECD and CD105-ECD (Mouse IgG, Monoclonal, BioLegend Cat. No. 303106, 343502, 304002, 307606, 921304, 328110, 344004, 323206). The expression of the various antigens was detected by flow cytometry.
6
Macrophage Immunophenotyping by Flow Cytometry
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As described above, macrophages were harvested using 1% (v/v) trypsin/EDTA (Life Technologies), washed with PBS and labeled with a master mix of fluorophore‐labeled human‐specific antibodies CD163‐FITC, CD80‐PE, HLA‐DR‐PE/Cy7, CD206‐APC (all BioLegend), CD14‐APC/Cy7 (BD Biosciences), and a Live/Dead violet fixable staining kit (Molecular Probes).[54 , 61 ] Samples were measured with FACS Canto II (BD Biosciences) and analyzed using FlowJo Version 8.8.6 (TreeStar Inc.). Surface marker expression levels were normalized to the unstimulated controls (set as 1).
7
Flow Cytometric Analysis of Adipose Macrophages
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The SVF pellets isolated from adipose tissue were washed with red blood cell lysis buffer (NH4Cl 150 mM, KHCO3 10 mM, Na2EDTA 0.1 mM, pH = 7.4) and PBS before staining with antibodies or the isotype control antibodies in PBS with 3% BSA for 1 hr at 4 °C. After washing with PBS, the cells were fixed in 4% formalin and analysed on LSR Fortessa Analyzer (BD Biosciences). Antibodies used are F4/80-FITC (1:100, Biolegend, #123108, BM8), CD11b-pacific blue (1:100, Biolegend, #101224, M1/70), CD206-APC (1:100, Biolegend, #141708, C068C2) and CD11c-PE (1:100, Biolegend, #117308, N418). The lymphocyte singlets were gated and adipose macrophages were defined as F4/80+ CD11b+ cells, followed by determination of macrophage subtypes.
8
Comprehensive BAL Immune Cell Profiling
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Cells collected from the BAL were surface stained with CD16/32 (Fc block; 2.4G2), LIVE/DEAD™ Fixable Blue Dead Cell Stain kit (ThermoFisher Scientific, Waltham, MA), CD11b PerCP/Cy5.5 (M1/70), Ly6G BV785 (1A8), SiglecF APC/Cy7 (E50-2440), F4/80 BV421 (T45-2342; BD Biosciences), CD200R PE (OX-110; BioLegend), CD11c PE/Cy7 (N418; BioLegend) and CD206 APC (C068C2; BioLegend) prior to fixation with BD Cytofix™ (BD Biosciences, San Jose, CA). Samples were collected on a Cytek Aurora managed by the United Flow Core of the University of Pittsburgh and data was analyzed using FlowJo V10 software (FLOWJO, LLC, OR).
9
Tumor Dissociation and Immune Cell Analysis
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Tumors were minced into thin pieces and were dissociated in DNase I (1 U/ml; Roche, Indianapolis, IN, USA), collagenase D (1 mg/ml; Roche), and 0.125% trypsin-EDTA (Gibco) in pre-warmed DMEM (Welgene) for an hour at 37 °C with gentle agitation. The tissues were mechanically dissociated on a 100 μm nylon mesh strainer. Next, the single cells were passed through a 40 μm nylon mesh strainer. Spleens were also mechanically dissociated on 40 μm nylon mesh strainer. RBCs were lysed for 5 minutes in 1X Pharmlyse buffer.
Cells were stained with fluorescently tagged antibodies. All data were detected by a FACSCalibur cytometric system and analyzed by FlowJo software. The following fluorophore-labeled antibodies were purchased from e-bioscience (San Diego, CA, USA): CD45-FITC, CD3-FITC, CD4-PE, CD4-FITC, CD8-APC, CD11b-APC, CD11c-PE, CD25-PE, CD25-APC, B220-FITC, CD19-PE, CD86-APC. Foxp3-Alexa Fluor647 was purchased from BD bioscience, and F4/80-PE and CD206-APC were obtained from Biolegend (San Diego, CA, USA).
Cells were stained with fluorescently tagged antibodies. All data were detected by a FACSCalibur cytometric system and analyzed by FlowJo software. The following fluorophore-labeled antibodies were purchased from e-bioscience (San Diego, CA, USA): CD45-FITC, CD3-FITC, CD4-PE, CD4-FITC, CD8-APC, CD11b-APC, CD11c-PE, CD25-PE, CD25-APC, B220-FITC, CD19-PE, CD86-APC. Foxp3-Alexa Fluor647 was purchased from BD bioscience, and F4/80-PE and CD206-APC were obtained from Biolegend (San Diego, CA, USA).
10
Tumor Macrophage Profiling via Flow Cytometry
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To prepare single-cell suspensions for flow cytometry, fresh tumor tissue was dissected into approximately 1 to 3 mm3 fragments and digested with 80 U/mL collagenase (Invitrogen) in DMEM containing 10% FBS for one hour at 37°C while shaking. After red blood cell (RBC) lysis, single-cell suspensions were filtered and incubated for 20 minutes on ice with the following antibodies (1:100): CD45-PE (eBioscience, San Diego, CA), F4/80-PE-Cy7 (eBioscience, San Diego, CA), CD11b-FITC (BD Biosciences San Jose, CA), CD206-APC (Biolegend, San Diego, CA),CD68-PerCP-Cy5.5 for macrophages (Biolegend, San Diego, CA), Cells were washed with phosphate-buffered saline (PBS) before analysis on the BD LSR-II flow cytometer (Beckman Coulter).
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