LPS (50 μg) was injected into the left footpad of the mice on day 0. PHC-NPs were injected subcutaneously into the footpad at 0.15 mg/time point. Injection time points are specified in the text below. At the terminal time point, mice were euthanized for footpad excision and caliper measurements. For evaluation of immune cell populations, footpad skin was collected, digested, and prepared as described in 2.10 . Cells were stained with AlexaFluor700 anti-CD45, PE anti-Ly6G, APC anti-F4/80, as well as FITC anti-CD206 (#141703, BioLegend), Pacific Blue anti-CD86 (#105021, BioLegend), or Brilliant Violet 421 anti-CD86 (#105031, BioLegend).
Anti cd206 fitc
Manufactured by BioLegend
Sourced in United States
Anti-CD206-FITC is a fluorescently labeled antibody that binds to the CD206 surface receptor, also known as the mannose receptor. CD206 is expressed on the surface of macrophages and dendritic cells. This product can be used for the identification and characterization of cell populations expressing CD206 by flow cytometry.
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Lab products found in correlation
Anti f4 80 apc, by Thermo Fisher Scientific (3 mentions)
Anti cd86 pe, by BioLegend (3 mentions)
Anti cd68 apc, by BioLegend (2 mentions)
Fitc anti cd16 32, by BioLegend (2 mentions)
Anti cd11c fitc, by BioLegend (2 mentions)
Anti cd11b pe, by BioLegend (2 mentions)
Anti cd4 fitc, by BioLegend (2 mentions)
Alexafluor700 anti cd45, by BioLegend (1 mentions)
Foxp3 buffer set, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Facsaria 3, by BD (1 mentions)
Anti cd45 bv421, by BioLegend (1 mentions)
Anti cd11b apc cy7, by BioLegend (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
7 aad, by BioLegend (1 mentions)
Pe cy7 anti f4 80, by BioLegend (1 mentions)
F4 80 brilliant violet 421, by BioLegend (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
13 protocols using anti cd206 fitc
1
Immune Cell Analysis in Footpad Inflammation
2
Profiling Surface Antigens in Macrophages
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Surface antigen expression (F4/80, iNOS, arginase 1, CD86, and CD206) on BMDMs/Raw 264.7 cells induced by RAP or IL-4 were determined by FCM. BMDMs and RAW264.7 cells were disassociated and collected by pipetting repeat. The, cells were washed with PBS once and stained with the following monoclonal antibodies diluted in 1% FBS in PBS: APC anti-F4/80 (eBioscience), FITC iNOS type II (BD Biosciences), mouse arginase 1 fluorescein-conjugated antibody (R&D system), FITC anti-CD206 (BioLegend), PE anti-CD86 (BioLegend). For intracellular staining, the Foxp3 buffer set (eBioscience) was used; the cells were fixed and permeated for 30 min. After staining, the cells were washed twice with PBS and analyzed by BD Bioscience FACS Canto II flow cytometry (BD Biosciences). The data were analyzed using FlowJo software. The amount of surface antigen expression was calculated according to the previous study [34 (link)].
3
Astragali Radix Polysaccharide Immunomodulation
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APC anti-F4/80, FITC iNOS type II, and mouse arginase 1 fluorescein-conjugated antibody were purchased from eBioscience, BD Biosciences, and R&D System, respectively. FITC anti-CD206 and PE anti-CD86 were bought from BioLegend (San Diego, CA, USA) SYBR® Select Master Mix for qRT-PCR amplification was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Monoclonal anti-Notch1, monoclonal cleaved Notch1, β -actin, anti-rabbit IgG HRP-linked antibody and anti-mouse IgG HRP-linked antibody were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Primers were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). γ-secretase inhibitors (DAPT and s2188) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
RAP was isolated from boiling water extract of Astragali Radix, followed by leaching, ethanol precipitation, dialysis, protein depletion, combination, and concentration. RAP used in this study is same to the one we used in our pervious study [16 (link)]. The endotoxin of RAP samples was detected by PYROGENT™ Plus gel clot LAL assay kit (Lonza, Cologne, Germany) according to the manufacturer’s instruction. No endotoxin contamination was found in RAP.
RAP was isolated from boiling water extract of Astragali Radix, followed by leaching, ethanol precipitation, dialysis, protein depletion, combination, and concentration. RAP used in this study is same to the one we used in our pervious study [16 (link)]. The endotoxin of RAP samples was detected by PYROGENT™ Plus gel clot LAL assay kit (Lonza, Cologne, Germany) according to the manufacturer’s instruction. No endotoxin contamination was found in RAP.
4
Poria cocos Modulates Macrophage Activation
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The Poria cocos sclerotium was obtained from Guangyintang Traditional Chinese Medicine Company (Anhui, China). Murine macrophage cell lines RAW 264.7 were gained from the Chinese Academy of Sciences. Beyotime Biotechnology (Shanghai, China) was the source of the dimethyl sulfoxide (DMSO), neutral red solution, and MTT powder. Lipopolysaccharide (LPS) was offered by Sigma-Aldrich, Shanghai, China). ELISA kits for the quantification of TNF-α, IL-10, IL-6, and IL-12, and the BCA kit were obtained from Jianglai Biotech Co. (Shanghai, China). The SPARKscript II RT Plus Kit for reverse transcription and the 2×SYBR Green qPCR Mix for amplification were bought from Cisco Biotech Co. (Binzhou, China). Primers were purchased from Sangon Biotech Co. (Shanghai, China). FITC anti-CD206 and PE anti-CD86 were supplied by BioLegend (San Diego, CA, USA). Primary antibodies against Notch1, Hes1, β-actin (Zhengneng Bio Co., Chengdu, China), Jagged1 (Boaosen Bio Co., Beijing, China), respectively, were used. All other reagents mentioned in this study were of analytical grade.
5
Immunophenotyping of Adipose Tissue Macrophages
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Flow cytometry was performed as described previously, with slight modification91 (link),102 (link). Cells in the stromal vascular fraction of eAT were stained with an anti-CD16/32 monoclonal antibody (TruStain fcX; Biolegend) to avoid non-specific staining and 7-AAD (Biolegend) to detect dead cells. The cells were further stained with the fluorescently labeled antibodies BV421–anti-CD45 (Biolegend, clone 30-F11), PE–anti-I-Ab MHC class II (Biolegend, clone AF6-120.1), FITC-anti-CD206 (Biolegend, clone C068C2), PE-Cy7–anti-F4/80 (Biolegend, clone BM8), and APC-Cy7–anti-CD11b (Biolegend, clone M1/70). Samples were analyzed by using BD FACSAria III (BD Biosciences), and data were analyzed by using FlowJo 9.9 (Tree Star, Ashland, Oregon, USA). The CD45+CD11b+F4/80+ cells were defined as the macrophage population91 (link). Among macrophages, the MHC II+/highCD206–/low cells and MHC II+CD206high cells were defined as the M1- and M2-like macrophage populations, respectively.
6
Lamina Propia Mononuclear Cell Profiling
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The lamina propia mononuclear cells were stained with fluorescence‐tagged primary antibodies (Brilliant Violet 421‐F4/80, Biolegend, San Diego, CA, USA; FITC‐anti‐CD11b, MACS, Bergisch Gladbach, Germany, FITC‐anti‐CD206, Biolegend, San Diego, CA, USA) for 45 min at 4°C. Flow cytometry was performed using a BD LSRFortessa™ cell analyzer (BD Biosciences, San Jose, CA, USA).
7
Treg-mediated Macrophage Polarization Dynamics
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Monocytes from BALB/c spleen samples were sorted using a Mouse Monocyte Isolation Kit (Miltenyi), co-cultured with Tregs at monocyte:Treg ratios of 2:0 and 2:1, and exposed to different stimuli to determine the effect of a miR-146a deficiency in Tregs on macrophage polarization. Macrophage type 1 (M1) cells were stimulated with lipopolysaccharide (LPS; 10 µg/ml; Solarbio) + interferon (IFN)-γ (20 ng/ml; PeproTech), and macrophage type 2 (M2) cells were stimulated with LPS (10 µg/ml; Solarbio)+IL-4 (50 ng/ml; PeproTech). After 72 h of co-culture, the cells from each group were harvested and labeled with anti-CD16/32-FITC (Catalog No.: 101305; BioLegend, USA), anti-CD206-FITC (Catalog No.: 141703; BioLegend, USA), and anti-CD68-APC (Catalog No.: 137007; BioLegend, USA) antibodies, and the ratios of various types of cells were detected using flow cytometry.
The levels of tumor necrosis factor (TNF)-α, IL-1β, and IL-10 in the supernatant of co-cultured monocytes and Tregs were determined using ELISA kits (DAKEWE) according to the manufacturer’s instructions.
The levels of tumor necrosis factor (TNF)-α, IL-1β, and IL-10 in the supernatant of co-cultured monocytes and Tregs were determined using ELISA kits (DAKEWE) according to the manufacturer’s instructions.
8
Multiparametric Flow Cytometry Analysis
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All fluorophore-conjugated antibodies were obtained from BioLegend: anti-CD206-FITC (Cat. #321103), anti-CD163-PE(Cat. #333606), anti-CD64-PE/Cy7(Cat. # 305022), anti-CD80-APC (Cat. # 305220), anti-HLA-DR-APC/Cy7 (Cat. # 307618), anti-CD16-BV421 (Cat. # 360723), anti-CD1a-PerCP(Cat. # 300130), anti-CD45-APC/Cy7 (Cat. # 368516), anti-CD4-APC (Cat. # 300552), anti-CD8-BV510 (Cat. # 301048), anti-CD3-BV42 (Cat. # 344834). Cells were stained for 1 hour at 4°C with 2 mg/ml Globulins Cohn fraction II, III (Sigma) to inhibit non-specific antibody binding. In all conditions, doublets and multiplets were excluded by forward scatter pulse width (SSC-W) vs. forward scatter pulse area (SSC-A) gating. Gating of positively stained cells was determined by fluorescence-minus-one (FMO) controls. Cells were analyzed using an 8-color MACSQuant 10 (Miltenyi Biotec) with three laser sources (405 nm, 488 nm, 635 nm) and FlowJo 9.8.1 (Treestar).
9
Phenotypic Analysis of M1 and M2 Macrophages
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Phenotype markers of macrophages were determined by flow cytometry analysis.
For M1 macrophage marker CD11c, 100 μl macrophage suspension at 106 cells/ml was added to 5 μl anti-CD11c-FITC (BioLegend, USA), followed by incubation at 37 °C for 30 min, centrifugation, two PBS washes, and flow cytometry analysis (BD, USA). Negative control staining was performed using the corresponding isotype control antibodies.
For M2 macrophage marker CD206, it was tested by intracellular immunofluorescent staining. Briefly, cells were fixed in 1 ml/tube Fixation Buffer (Beyotime, China) in the dark for 15 min at room temperature. Then, cells were permeabilized in 1 ml/tube Permeabilization Buffer Triton X-100 (Beyotime, China) for 10 min at room temperature. Finally, 100 μl fixed/permeabilized macrophage suspension at 106 cells/ml was added to 5 μl anti-CD206-FITC (BioLegend, USA), followed by incubation at 37 °C for 30 min, centrifugation, two PBS washes, and flow cytometry analysis (BD, USA). Negative control staining was performed using the corresponding isotype control antibodies.
For M1 macrophage marker CD11c, 100 μl macrophage suspension at 106 cells/ml was added to 5 μl anti-CD11c-FITC (BioLegend, USA), followed by incubation at 37 °C for 30 min, centrifugation, two PBS washes, and flow cytometry analysis (BD, USA). Negative control staining was performed using the corresponding isotype control antibodies.
For M2 macrophage marker CD206, it was tested by intracellular immunofluorescent staining. Briefly, cells were fixed in 1 ml/tube Fixation Buffer (Beyotime, China) in the dark for 15 min at room temperature. Then, cells were permeabilized in 1 ml/tube Permeabilization Buffer Triton X-100 (Beyotime, China) for 10 min at room temperature. Finally, 100 μl fixed/permeabilized macrophage suspension at 106 cells/ml was added to 5 μl anti-CD206-FITC (BioLegend, USA), followed by incubation at 37 °C for 30 min, centrifugation, two PBS washes, and flow cytometry analysis (BD, USA). Negative control staining was performed using the corresponding isotype control antibodies.
10
Multiparametric Flow Cytometry of Macrophages
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Bone marrow‐derived macrophages were stained with the following fluorescently labeled mAbs: anti‐CD206 FITC, anti‐CD11b PE, anti‐CD45 Alexa Fluor 700, anti‐F4/80 allophycocyanin, (all from BioLegend). The cells were stained with Live Dead Stain Aqua (Life Technologies) or calcein‐AM (Thermofisher) prior to surface staining. Samples were acquired on a BD LSRII flow cytometer (BD Biosciences).
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