Fig. 1

Phenotypic characterization of LPS- and IL-10-stimulated macrophages derived from human CD14⁺ peripheral blood monocytes. a Representative images of actin and tubulin stainings of LPS- and IL-10-stimulated macrophages polarized in absence of other external stimuli (mac) or in the presence of 10ng/ml LPS (LPSmac) or IL-10 (IL-10mac), respectively. F-actin was stained with Phalloidin-FITC (green), α–tubulin with a specific monoclonal antibody followed by incubation with AlexaFluor594 secondary antibody (red) and nuclei were counterstained with DAPI (blue). Scale bars represent 50 μm. b Morphological differences between macrophage populations were quantified by calculating the cell aspect ratio (quotient between cell major and minor axes) of actin/tubulin stained cells. Chart reflects measurements of at least 100 cells per donor from, at least, 3 distinct donors. Bars represent mean values and flags indicate standard deviations. c Cytokine production profile of LPS- and IL-10-stimulated macrophages. Cytokine concentration was measured by ELISA in conditioned media from distinct macrophage populations. Charts indicate fold increase in IL-6, IL-10 and TNF-α expression, in comparison to unstimulated macrophages. Data is representative of the cytokine profile of cells derived from at least 7 different donors. Bars represent mean values and flags indicate standard deviations. d Expression of typical macrophage lineage (CD14) and polarization markers (HLA-DR and CD163) was determined by flow cytometry of unstimulated, LPS- and IL-10-stimulated macrophages. Scatter charts represent percentage of positive cells for each cell surface marker considering data obtained with cells derived from 5 different donors. *, significantly different at p < 0.05. IL-10, interleukin-10; LPS, lipopolysaccharide