M0M-CSF (2×106/mL) were treated with CS (0.2 µM) or vehicle (0.1% DMSO) for 48 hours. Then, cells were stained in PBS pH 7.4 containing 0.5% bovine serum albumin, 2 mM EDTA, and 0.1% sodium azide by Zombie Aqua™ Fixable Viability Kit (Biolegend, San Diego, CA) for 5 minutes at 4°C to determine cell viability. Non-specific antibody binding was blocked by using mouse serum (10 minutes at 4°C) prior to staining by the following fluorochrome-labeled antibodies (20 minutes, 4°C): FITC anti-human CD14 (clone M5E2, #555397, BD Biosciences, San Jose, CA), APC-H7 anti-human CD80 (clone L307.4, #561134, BD Biosciences), PE-Cy7 anti-human CD54 (clone HA58, #353115, Biolegend), PE anti-human CD163 (clone GHI/61, #556018, BD Biosciences), and APC anti-human CD206 (clone 19.2, #550889, BD Biosciences) to determine M1 and M2 surface marker expression using a LSRFortessaTM cell analyzer (BD Biosciences), and data were analyzed using FlowJo X Software (BD Biosciences).
Apc anti human cd206 clone 19
Manufactured by BD
Sourced in United States, Germany
APC anti-human CD206 (clone 19.2) is a fluorophore-conjugated antibody that binds to the human CD206 (Mannose Receptor) protein. CD206 is a cell surface receptor expressed on certain immune cells, such as macrophages and dendritic cells. This antibody can be used in flow cytometry and other immunoassays to identify and study CD206-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Fitc anti human cd14 clone m5e2, by BD (3 mentions)
Apc h7 anti human cd80 clone l307, by BD (3 mentions)
Pe cy7 anti human cd54 clone ha58, by BioLegend (3 mentions)
Pe anti human cd163 clone ghi 61, by BD (3 mentions)
Zombie aqua fixable viability kit, by BioLegend (3 mentions)
Lsrfortessa cell analyzer, by BD (3 mentions)
Rainbow calibration particles, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
4 protocols using apc anti human cd206 clone 19
1
Macrophage Polarization Analysis
2
Macrophage Immunophenotyping and Viability
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Macrophages were stained in PBS pH 7.4 containing 0.5% BSA, 2 mmol/L EDTA and 0.1% sodium azide by Zombie Aqua™ Fixable Viability Kit (Biolegend, San Diego, CA, USA) to determine cell viability (5 min, 4 °C) and by fluorochrome-labelled antibodies (20 min, 4 °C): FITC anti-human CD14 (clone M5E2, #555397, BD Bioscience, San Jose, CA), APC-H7 anti-human CD80 (clone L307.4, #561134, BD Bioscience), PE-Cy7 anti-human CD54 (clone HA58, #353115, Biolegend), PE anti-human CD163 (clone GHI/61, #556018, BD Bioscience), APC anti-human CD206 (clone 19.2, #550889, BD Bioscience) to determine M1 and M2 surface marker expression using a LSRFortessa™ cell analyzer (BD Bioscience), and data were analyzed using FlowJo X Software (BD Bioscience)21 (link).
3
M1/M2 Macrophage Polarization Assay
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M0M-CSF MDM were treated with TWG (1 µg/mL) or vehicle (0.1% DMSO) for 48 h. Then, cells were stained in PBS pH 7.4 containing 0.5% BSA, 2 mM EDTA and 0.1% sodium azide by Zombie Aqua™ Fixable Viability Kit (Biolegend, San Diego, CA, USA) for 5 min at 4 °C to determine cell viability. Non-specific antibody binding was blocked by using mouse serum (10 min at 4 °C) prior to staining by the following fluorochrome-labelled antibodies (20 min, 4 °C): FITC anti-human CD14 (clone M5E2, #555397, BD Bioscience, San Jose, CA, USA), APC-H7 anti-human CD80 (clone L307.4, #561134, BD Bioscience), PE-Cy7 anti-human CD54 (clone HA58, #353115, Biolegend, Koblenz, Germany), PE anti-human CD163 (clone GHI/61, #556018, BD Biosciences, Heidelberg, Germany), APC anti-human CD206 (clone 19.2, #550889, BD Bioscience) to determine M1 and M2 surface marker expression using LSRFortessaTM cell analyzer (BD Bioscience), and data were analyzed using FlowJo X Software (BD Bioscience).
4
Multiparametric Flow Cytometry Analysis
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Flow cytometry was performed as initially described with minor modifications. 20 A total of 100,000 stroma-vascular cells were incubated for 30 minutes at 4 C in phosphatebuffered saline supplemented with 0.5% bovine serum albumin and 2 mmol/L EDTA that contained the following fluorescent labeled antibodies: Brilliant Violet 510 antihuman CD45 Antibody (clone HI30, dilution 1/20, Biolegend, San Diego, CA), APC anti-human CD206 (clone 19.2, dilution 1/10, BD Biosciences), and PE-Vio 770 anti-human CD14 (clone TÜK4, dilution 1/50, Miltenyi Biotec). As control, a panel of appropriate isotype antibodies was used. The labeled cells were washed with phosphate-buffered saline and analyzed in a FACS Canto II flow cytometer using Diva Pro software version 9 (BD Biosciences). Cell debris, dead cells, and doublets were excluded based on scatter signals. Compensation was established using compensation particles set (BD Biosciences) and single staining, and flow cytometer calibration was performed with rainbow calibration particles (BD Biosciences).
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