Amicon ultra 15 centrifuge filter unit

Exosomes were prepared from neuronal primary culture NCM. The medium was collected and centrifuged at 300×g for 10 min and 2000×g for 10 min at 4 °C. Following centrifugation, the supernatant was passed through a 0.22-μm sterile filter (Steritop™ Millipore, MA, USA). Then, the filtered supernatant was transferred to the upper compartment of an Amicon Ultra-15 Centrifuge Filter Unit (Millipore) and centrifuged at 4000×g until the volume of the upper compartment was reduced to ~ 200 μL. The liquid was loaded onto the top of a 30% sucrose/D2O cushion in a sterile Ultra-Clear™ tube (Beckman Coulter, CA, USA) and underwent a 10,000×g centrifugation step for 60 min at 4 °C in an Optima L-100 XP Ultracentrifuge (Beckman Coulter) in order to purify the exosomes. Partially purified neuron-derived exosomes were recovered using an 18-G needle diluted in PBS and centrifuged at 4000×g at 4 °C in a filter unit until the final volume reached 200 μL. Exosomes were used for downstream experiments or stored at − 80 °C
A Nanosight LM10 System (Nanosight Ltd., Navato, CA) was used to analyze the distribution of vesicle diameters from the exosomes. A transmission electron microscope (TEM; Tecnai 12; Philips, Best, The Netherlands) was used to observe the morphology of the acquired exosomes. Western blotting was used to determine specific exosome surface markers, such as CD9, CD63, and CD81.

HDL were isolated from 600 μl of serum by immunoprecipitation using goat anti-apoA-I beads as previously described. Briefly, human serum was applied to a column containing goat anti-human apoA-I antibody covalently coupled to Cyanogen bromide (CNBr)-activated Sepharose 4B (Amersham Pharmacia Biotech). To remove proteins non-specifically bound to the beads, the column was washed 10 times with 1X Tris Buffered Saline (TBS). HDL that bound to the column were eluted with stripping buffer (0.1 M acetic acid) and neutralized immediately with 1 M Tris, pH 11 (final concentration, 0.11 M). Samples were further concentrated using Amicon Ultra-15 centrifuge filter unit (Millipore, Catalogue # UFC901024) and Amicon Ultra-0.5, ultracel-10 membrane (Millipore, Catalogue # UFC501096).

MSCs were incubated for 48 h after reaching 80% confluency, replacing the media with exosome-depleted FBS. Then, the supernatant was collected and centrifuged successively at 300 × g for 10 min and 2,000 × g for 10 min at 4 °C. After centrifugation, the cell supernatant was filtered through a 0.22-μm sterile filter (Steritop™ Millipore, Burlington, MA) and applied to the upper compartment of an Amicon Ultra-15 Centrifuge Filter Unit (Millipore). Then the supernatant was centrifuged at 4,000 × g till the volume decreased to 200 μl in the upper compartment. The ultra-filtered supernatant was then washed twice with PBS and re-filtered to another 200 μl. The liquid was loaded onto the top of a 30% sucrose/D2O cushion in a sterile Ultra-Clear™ tube (Beckman Coulter, Asphalt, CA, USA), followed by centrifugation at 100,000 × g for 60 min at 4 °C with an optima L-100 XP Ultracentrifuge (Beckman Coulter). Exosomes were stored at -80 °C.