We used mouse J774 cells, non-obese diabetic, severe combined immunodeficient, common gamma chain knockout mouse (NOD-SCID-Gamma, or NSG) macrophages, and human macrophages for our experiments. J774 macrophages were activated 24 hours before phagocytosis assays using 100 ng/ml recombinant mIFNγ (eBioscience). Cancer cells were either GFP+ or labelled with carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and were incubated with 10 μg/ml of CD47 blocking reagents, isotype controls, or tumour-targeting antibodies for 30 minutes. Macrophages were then co-cultured in non-adherent plates with cancer cells at a 1:1 (J774) or 1:2 (NSG, human macrophages) ratio. Phagocytosis was analysed by flow cytometry after 2 hours. Mouse macrophages were identified with PE/Cy7- or allophycocyanin (APC)-anti-mouse F4/80 (Biolegend), and human macrophages were identified with APC-anti-human CD206 (Biolegend). Phagocytosis was quantified as the percentage of F4/80+ or CD206+ cells that engulfed CFSE+/GFP+ tumour cells per total F4/80+ or CD206+ population. For soluble factor assays, Raji cells and J774 macrophages were suspended in supernatant harvested from cultured M14 melanoma cells titrated with new IMDM medium for the duration of the assay.
Apc anti human cd206
Manufactured by BioLegend
Sourced in United States
APC anti-human CD206 is a fluorescently-labeled antibody used for the detection and analysis of the CD206 receptor expressed on the surface of certain immune cells, such as macrophages, dendritic cells, and monocytes. It is a useful tool for identifying and studying these cell populations in flow cytometry applications.
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Lab products found in correlation
Pe anti human cd86, by BioLegend (6 mentions)
Apc anti human cd86, by BioLegend (5 mentions)
Facscalibur, by BD (3 mentions)
Pe anti human cd206, by BioLegend (3 mentions)
Cellquest software, by BD (3 mentions)
Staining buffer, by BD (3 mentions)
Alexa fluor 488 anti human cd11b, by BioLegend (3 mentions)
Accutase, by Nacalai Tesque (3 mentions)
Human trustain fcx fc receptor blocking solution, by BioLegend (3 mentions)
Apc anti mouse f4 80, by BioLegend (2 mentions)
Recombinant mifnγ, by Thermo Fisher Scientific (2 mentions)
Fitc anti human cd34, by BioLegend (2 mentions)
Pe anti human cd45, by BioLegend (2 mentions)
Alexa fluor 488 anti human cd105, by BioLegend (2 mentions)
Fitc anti human cd14, by BioLegend (2 mentions)
Pe anti human cd163, by BioLegend (2 mentions)
Facscanto 2, by BD (2 mentions)
Apc anti human cd11b, by BioLegend (1 mentions)
Pe anti human cd80, by BioLegend (1 mentions)
Apc anti mouse cd206, by BioLegend (1 mentions)
14 protocols using apc anti human cd206
1
Macrophage-mediated Phagocytosis of Tumor Cells
2
Immunophenotyping of GMSCs and Macrophages
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The expression of CD73, CD90, CD105, CD34, CD45, and CD11b in GMSCs and CD11b, CD206, and CD86 in PBMC-derived macrophages were analyzed using the FACS Calibur (Becton Dickinson, USA) and the CellQuest software (Becton Dickinson). The adherent cells were washed with PBS and collected using Accutase (Nacalai Tesque) and resuspended in 50 μL staining buffer (BD Pharmingen, country). The harvested cells were blocked with 2 μL Human Trustain FcX (Fc receptor Blocking Solution; BioLegend) for 10 min at room temperature, and stained with 2 μL antibodies namely, FITC anti-human CD73, FITC anti-human CD90, Alexa Fluor® 488 anti-human CD105, FITC anti-human CD34, PE anti-human CD45, FITC anti-human CD11b, Alexa Fluor® 488 anti-human CD11b, PE anti-human CD206, APC anti-human CD206, PE anti-human CD86 and APC anti-human CD86 (BioLegend) in the dark for 30 min at 4 °C.
3
Macrophage Phenotype Analysis by Flow Cytometry
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The tMACs or iMACs were stimulated with LPS and IFN-γ for the indicated time. The single-cell suspensions were then prepared and incubated with an antibody or antibody cocktails for 15 min at room temperature for cell surface staining. Antibodies used in this study were PE Mouse IgG1, κ isotype (Biolegend, Cat: 400113, Clone: MOPC-21, Lot: B245984), APC Mouse IgG1, κ isotype (Biolegend, Cat: 400119, Clone: MOPC-21, Lot: B243042), FITC Mouse IgG1, κ isotype (Biolegend, Cat: 400107, Clone: MOPC-21, Lot: B199152), APC anti-human CD206 (Biolegend, Cat: 321109, Clone: 15-2, Lot: B348965), APC anti-human CD86 (Biolegend, Cat: 305411, Clone: IT2.2, Lot: B351349), PE anti-human CD80 (Biolegend, Cat:305208, Clone: 2D10, Lot: B330518), PE anti-human CD163 (Biolegend, Cat: 333606, Clone: GHI/61, Lot: B347256), FITC anti-human CD14 (Biolegend, Cat: 325604, Clone: HCD14, Lot: B268830) and APC anti-humanCD11B (Biolegend, Cat: 301309, Clone: ICRF44, Lot: B278346). These antibodies were used at a 1:100 dilution. Data were recorded on Beckman DxFLEX and analyzed with the FlowJo V10 software.
4
Multiparametric Flow Cytometric Analysis of Immune Cell Phenotypes
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For surface staining, cells were washed and incubated with FITC-anti-human CD14 (BioLegend), PE-anti-human CD197 (BioLegend), APC-anti-human CD206 (BioLegend), or PerCP-5.5-anti-mouse F4/80 (BioLegend), PE-anti-mouse CD11b (BioLegend), APC-anti-mouse CD206 (BioLegend) in PBS containing 2% FBS at 4°C for 30 min. Isotype-matched immunoglobulin served as controls. For intracellular staining, cells were fixed with fixation buffer (BD Biosciences) for 30 min at 4°C. The fixed cells were permeabilized with the permeabilization solution (BD Biosciences) at room temperature for 30 min. Cells were incubated overnight with Alexa Fluor 488-anti-mouse iNOS (eBioscience) at 4°C. Then, labeled cells were sorted by using the Aria III cell sorter (BD Biosciences) or analyzed by using BD FACSCanto II (BD Biosciences) with FACSDiva software.
5
Phenotyping M1, M2, and M0 Macrophages
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Flow cytometric analysis of M1-, M2-, and M0-MΦs before and after infection with Mtb was done per published procedures. MΦs were stained and analyzed on the Fortessa flow cytometer (Beckton Dickinson), and the data were processed using FlowJo v10 software (Tree Star, Inc.). Fluorochrome-conjugated antibodies used for flow cytometry were as follows: PE-Cy7 anti-human CD68 (BD Biosciences, cat no. 565595; clone: Y1/82A), PE anti-human CD14 (Invitrogen, cat no. 12-0149-42; clone: 61D3), APC anti-human CD206 (BioLegend, cat no. 321110; clone: 15-2), AF700 anti-human CD80 (BD Biosciences, cat no. 561133; clone: L307.4). Dead cells were excluded by using aqua fluorescent reactive dye (Invitrogen, cat no. L34957). GraphPad PRISM used for data analysis. Other antibodies are listed in Supplementary Tables.
6
Immunophenotyping of M1/M2 Macrophages
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Flow cytometry was used to detect the surface markers of THP-1 cells that had been stimulated for 24 hours and collected from 6-well plates. The suspension, which had 1 × 106 M1-polarized and M2-polarized cells, was then divided into 1.5 ml EP tubes and incubated with the following antibodies (PE anti-human CD86 and APC anti-human CD206; both from BioLegend, San Diego, CA, USA) on ice for 30 min in the dark. Flow cytometry (BD Biosciences, San Diego, CA, USA) was used to detect cells that had been suspended in 500 μl PBS with 3% FBS after being washed twice.
7
Flow Cytometric Immunophenotyping of Cells
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Cells were collected and washed twice with phosphate-buffered saline (PBS). The cells were then pre-incubated with human TrustainFcXTM (Fc receptor blocking solution, Cat. No. 422301; BioLegend, San Diego, CA) for 10 min at room temperature. Cells were stained with antibody for 20 min on ice before analysis using a flow cytometer (FC500; Beckman, Shanghai, China). Flowjo V10 software was used for data analysis. All experiments were repeated three times. The antibodies used are as follows: PE anti-human CD163 (Cat. No. 333605; BioLegend), APC anti-human CD206 (Cat. No. 321109; BioLegend), and PE anti-human CXCR4 (Cat. No. 306505; BioLegend).
8
Polarization of THP-1 Monocytes to M1 and M2 Macrophages
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THP-1, the most widely used model for the human monocytes/macrophages, was obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China), and routinely cultured in RPMI 1640 medium (HyClone, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. We polarized THP-1 monocytes according to the procedure reported previously [25 (link)]. THP-1 were firstly differentiated into M0 macrophages with 100 ng/ml phorbol 12-myristate 13-acetate (PAM, Sigma, USA) for 24 h. M0 cells were then polarized into M1 macrophages with incubation with 100 ng/ml LPS (PeproTech, USA) and 20 ng/ml IFN-γ (PeproTech) for an additional 48 h. To generate M2 phenotype, M0 populations were exposed to 20 ng/ml IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) for another 48 h. The phenotypes of polarized macrophages were determined by cell makers via flow cytometry. M1 and M2 macrophages were stained using lineage-specific antibodies, with PE anti-human CD86 for M1, APC anti-human CD206 (BioLegend) for M2, and FITC anti-human CD11b (BioLegend) for all monocytes. FlowJo software was used for statistical analysis.
9
Immunophenotyping of GMSCs and Macrophages
10
Flow Cytometry Analysis of Macrophage Phenotypes
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NPC cells were co-cultured with macrophages for 48 h. Then the macrophages were trypsinized and resuspended in phosphate buffer saline to a concentration of 1 × 107/ml, and dispensed into 100 μl into the flow cytometry tubes. Overall, 5 μl extracellular antibody APC antihuman CD206 (321109, Biolegend, CA, USA) was added and kept still for at least 30 min, then 5 μl intracellular antibody FITC antihuman CD68 (333805, Biolegend, CA, USA), and PE antihuman CD86 (374205, Biolegend, CA, USA) were incubated in the dark for at least 30 min after permeabilization. Then protein expression was detected by flow cytometry (Beckman Coulter, Brea, CA, USA).
The xenografts were cut and dispersed into single cells, filtered through a 300-mesh filter cloth and centrifuged. The cells were resuspended in PBS to a concentration of 1 × 107/ml, and dispensed into 100 μl into the tubes. The following steps were the same as the former cell experiments.
The xenografts were cut and dispersed into single cells, filtered through a 300-mesh filter cloth and centrifuged. The cells were resuspended in PBS to a concentration of 1 × 107/ml, and dispensed into 100 μl into the tubes. The following steps were the same as the former cell experiments.
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