Cd206

CD14, CD68, CD11b, CD11c, CD83 and CD1a fluorochrome-conjugated antibodies (BD Biosciences) were used to stain the immature macrophages in the dark at 4 °C for 15 min. After co-culture experiments for 72 h, immature macrophages were obtained by incubating with CD163 (Miltenyi Biotec), CD204 (Miltenyi Biotec), CD206 (Miltenyi Biotec) and B7-H1 (eBioscience) fluorochrome-conjugated antibodies in the dark at 4 °C for 15 min, followed by incubation with iNOS antibody (Abcam) and fluorescently-labeled secondary antibody. Cells were analyzed on a FACScan flow cytometer (BD Biosciences), and data were interpreted using the Flowjo software (Tree Star). Each experiment was performed in triplicate.

Flow cytometry was performed as described (40 (link)). Mf were resuspended with FACS buffer (PBS supplemented with 0.2% BSA, 0.01% NaN₃) and stained with PE-conjugated mouse anti-human mAbs against CD14, CD80, CCR7, and TREM-1 (clone 193015, obtained from BioLegend, Milano, Italy), CD68 (obtained from Dako, Milano), CD206 (obtained from Miltenyi Biotec), CD86, HLA-DR, CD36 (BD-Pharmingen, Milano, Italy), and isotype-matched IgG (obtained from BioLegend) for 30 min at 4°C, after preincubation with rabbit IgG (obtained from Sigma) to block non-specific sites. Four-color flow cytometric analysis was carried out to analyze SFMCs using the following Abs: anti-CD68-FITC (obtained from Dako), anti-CD80-PE/Cy7, anti-CD206-APC, and anti-TREM-1-PE Abs (obtained from Biolegend). Fluorescence was quantitated on a FACSCalibur flow cytometer equipped with CellQuest software (BD-Biosciences). Cells were gated according to their light-scatter properties to exclude cell debris.