Eclipse e800m fluorescence microscope

EVs were labelled using the PKH26 Red Fluorescent Cell Linker kit (Sigma-Aldrich) according to the manufacturer’s instructions with minor modifications. Two microgram (2 μg) of the PKH26 labelled EVs, or the same volume of the PKH26-PBS control, were resuspended in Endothelial Cell Growth Medium with 10% FDE and added to 1 × 10⁴ HUVEC cells maintained at 37 °C in a humidified atmosphere with 5% CO₂. All samples were ultracentrifuged at 110,000× g for 1 h at 4 °C before being added to the cells and unincorporated dye from exosome labelling reactions was removed by using Exosome Spin Columns (MW 3000) (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After 24 h of incubation, uptake was stopped by washing and fixation in 3.7% PFA for 10 min. Cells were then stained with a fluorescein isothiocyanate (FITC)-conjugated phalloidin (Sigma-Aldrich) and visualized with a Nikon Eclipse E800M fluorescence microscope (Nikon, Tokyo, Japan).

Fragaria-derived EPDENs were stained with PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich), according to the manufacturer’s instructions, with minor modifications, as previously described [22 (link)]. Unincorporated dye from EPDEN labeling reactions was removed using Exosome Spin Columns (MW 3000) (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. PKH26-labeled EPDENs (2 µg/L × 10⁴ cells), or the same volume of the PKH26-PBS control, were added to semiconfluent ADMSCs. After 4 h and 24 h of incubation, uptake was stopped by washing and fixation in 3.7% PFA for 10 min. Cells were then stained with a FITC-conjugated phalloidin (Sigma-Aldrich) and visualized with a Nikon Eclipse E800M fluorescence microscope (Nikon, Tokyo, Japan).

Human OC precursors (PBMC) were isolated from buffy-coats of at least two healthy volunteers as previously described, and seeded in eight-well chamber slides. After 2 h of incubation at 37°C in a humidified 5% CO₂ atmosphere, the medium was replaced, and the resulting mononuclear adherent precursors were cultured for 7 days with collected CM at a defined ratio depending on the specific experiment (see Table 1A), and compared with the respective negative control.
For all the cell cultures, medium was changed every 3 or 4 days. When specifically mentioned, anti-IL-6 monoclonal antibody (TCZ, 100 μg/ml, Roche) or anti-IL-8 antibody (anti-IL-8 Ab, 5 μg/ml, R&D Systems) were added every 24 h. After 7 days of culture, cells were analyzed for TRACP expression (Acid Phosphatase, Leukocyte kit, Sigma-Aldrich), according to the manufacturer’s protocols, dark incubated with 2.25 μg/ml of Hoechst 33258 (Sigma-Aldrich) at RT for 10 min, and visualized with a Nikon Eclipse E800M fluorescence microscope (Nikon). TRACP-positive cells that showed more than three nuclei were considered as OC and counted. For assays, at least four replicates were performed.