EPDENs Uptake by ADMSCs Visualization

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).