Exosome Isolation and Characterization from HSCs

Exosomes were removed from FBS by serial ultracentrifugation [20 ] prior to using it for HSC culture. Exosomes were isolated from conditioned medium of P6 HSC using standardized steps of low and ultra-speed centrifugation [20 ]. Nanoparticle tracking analysis (Nanosight^TM, Malvern Instruments, Westborough, MA) was used to determine exosome size and frequency. Exosomes were further evaluated for morphology and size using a Tecnai G2 F20 cryogenic transmission electron microscope (FEI, Hillsboro, Oregon) as described [17 (link)]. For some experiments, HSC were treated with 500nM SYTO RNASelect™ Green Fluorescent Cell Stain (Thermo Fisher) for 12 hrs and then incubated in fresh medium (exosome-free) for 48 hrs prior to exosome isolation. For other experiments, miR-199a-5p mimic (Qiagen) was labeled with Cy3 dye using a Label II® miRNA labeling kit (Mirus Bio LLC, Madison WI) and then transfected into exosomes by electroporation using a Nucleofector kit (Lonza, Koln, Germany); exosomes were then re-purified using a PureExo kit (101Bio, Palo Alto, CA).