Exosome Uptake by Oligodendrocyte Progenitor Cells

To confirm the uptake of exosomes into OPCs, exosomes were labeled using a PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) according to the manufacturer’s protocol with minor modifications as previously described [42 (link)]. Three hundred microliters of each exosome suspension was mixed with 100 μL of Diluent C. Stain solution was prepared by adding 1.4 μL of PKH67 to another 300 μL of Diluent C. The exosome solution and stain solution were mixed, incubated at room temperature for four minutes, and the labeling reaction was stopped by adding 700 μL of FBS to the mixed solution. Labeled exosomes were added to OPCs and incubated at 37 °C for 24 hours. After the incubation, cells were fixed with 2% PFA solution. OPCs were immunostained using an anti-CD68 antibody (KP1, Dako, CA, USA) labeled with Alexa Fluor 594 goat anti-mouse IgG (A11005, Invitrogen) and observed using an Axioskop 2 plus fluorescent microscope (Carl Zeiss, Jena, Germany).