Phenotypic and Functional Characterization of Macrophages

After incubation and treatment with 1 µM RA, 5μg/ml CHO, 2.5 μg/ml NP3 (RA)-CHO, 2.5 μg/ml NP4-RA, 2.5 μg/ml NP8-CHO, 1.2 μg/ml abIL-10R, and 2.5 μg/ml NP3 (RA)-CHO+1.2 μg/ml abIL-10R for 48 hours, RAW 264.7 cells (1.5 × 104 cells/well in a 12-well plate) were collected with a cell scraper and blocked with 0.5 % BSA in PBS for 45 minutes. To evaluate whether the cells show characteristics of M1 or M2 macrophages, RAW 264.7 cells were labelled with anti-mouse CD68-FITC (1:1000) or CD163-PerCP antibody (1:1000), respectively [11] (link). Next, staining of intracellular IL-10 was performed. For this purpose, cells were fixed in 2% paraformaldehyde (PFA, BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with GolgiPlug™) for 15 minutes at room temperature. Next, cells were washed and permeabilized with 0.1% Triton X-100 for 5 minutes and, subsequently, blocked by 0.5% BSA in PBS for 45 minutes. For intracellular staining, permeabilized cells were incubated using a cytofix/cytoperm kit (BD Biosciences) for 1 h. The anti-mouse IL-10-FITC antibody (1:1000) was applied for 60 minutes at 4°C for intracellular IL-10 staining. After a final washing step, the cells were analysed by flow cytometry [11] (link).