Macrophage Immunophenotyping by Flow Cytometry

Joint macrophages were prepared and filtered with a cell strainer. F4/80 macrophages were used as pan-macrophage markers, iNOS was used as a marker of M1 macrophages and CD206 was used as a marker of M2 macrophages [18 (link),31 (link),32 (link)]. Surface staining was performed using the following monoclonal antibodies: anti-F4/80 FITC (eBioscience), anti-CD206 PE (BioLegend, San Diego, CA, USA). Anti-iNOS allophycocyanin was purchased from BD Biosciences. For intracellular staining of iNOS, Cell Stimulation Cocktail (a cocktail of phorbol 12-myristate 13-acetate, ionomycin, brefeldin A and monensin from eBioscience) was added and cultured for the last 5 hours before flow cytometric analysis as previously described [25 (link)].

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Ye L., Wen Z., Li Y., Chen B., Yu T., Liu L., Zhang J., Ma Y., Xiao S., Ding L., Li L, & Huang Z. (2014). Interleukin-10 attenuation of collagen-induced arthritis is associated with suppression of interleukin-17 and retinoid-related orphan receptor γt production in macrophages and repression of classically activated macrophages. Arthritis Research & Therapy, 16(2), R96.

Publication 2014

anti-F4/80 FITC anti-CD206 PE Cell Stimulation Cocktail brefeldin A monensin

Allophycocyanin Brefeldin a Cell Fitc Flow cytometric Inos Intracellular Ionomycin Joint Macrophages Monensin Monoclonal antibodies Phorbol myristate acetate

Corresponding Organization :

Other organizations : Shenzhen University

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Variable analysis

independent variables

Preparation and filtering of joint macrophages using a cell strainer
Use of F4/80 as a pan-macrophage marker
Use of iNOS as a marker of M1 macrophages
Use of CD206 as a marker of M2 macrophages
Surface staining using anti-F4/80 FITC, anti-CD206 PE, and anti-iNOS allophycocyanin antibodies
Intracellular staining of iNOS using Cell Stimulation Cocktail (containing phorbol 12-myristate 13-acetate, ionomycin, brefeldin A, and monensin) for the last 5 hours before flow cytometric analysis

dependent variables

Proportions of F4/80+, iNOS+, and CD206+ macrophage subpopulations

control variables

Not explicitly mentioned

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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