Isolation of NPC exosomes from cell culture supernatants and quality control of exosome preparations. A) Summary of the experimental procedure used for exosome purification. B) Negative staining electron microscopy of exosomes purified from NAD+C15 conditioned culture medium. Scale bar: 100 nm. Exosomes are characterized by a diameter of 50 to 100 nm and a frequent plate-like morphology. C) Western blot analysis of CD63 and gp96 in whole cell (CELLS) and exosome (EXO) protein extracts (NAD+C15). Regardless of the cell background, the CD63 tetraspanin is generally very abundant in exosomes. In contrast gp96 which is a cytoplasmic membrane protein is absent or at a very low concentration. Staining with anti-β-actin was used for loading control (although it is less abundant in exosomes than in whole cell extracts). Overall these data confirm the good quality of our exosome preparations.