Amicon ultra 15 centrifugation filters

AAV was produced in AAVpro(R) 293T cells (Takara, Kyoto, Japan; #632273) by co-transfection of the genome pAAV, an AAV helper plasmid, and pUCmini-iCAP-PHP.eB. pUCmini-iCAP-PHP.eB was a gift from Viviana Gradinaru (http://n2t.net/addgene:103005; RRID: Addgene 103005) (Chan et al., 2017 (link)). The AAV particles were precipitated with polyethylene glycol (PEG 8000, Sigma-Aldrich, St. Louis, MO; Cat# P2139-500G) and purified on an iodixanol gradient (OptiPrep, Sigma-Aldrich, Cat# D1556-250ML) with ultracentrifugation for 2.5 hr at 350,000 × g at 18°C. We filtered and concentrated the virus in PBS using Amicon Ultra-15 centrifugation filters (Millipore, Burlington, MA; #UFC905024). The viral titer was measured with the AAVpro(R) Titration Kit (Takara, #6233) and AAV was stored single-use aliquots at –80°C until injection.

After CSC exposure, 100 mL of cell supernatant was centrifuged at 3000 g for 15 min at 4 °C for debris removal. The clear media was subjected to further cleaning by filtration through 0.22 μm sterile filter (Millipore, MA, USA) to remove any remaining debris. The filtered media was transferred to Amicon^® Ultra-15 centrifugation filters (Millipore, Billerica, MA, USA) and centrifuged at 2500 g for 35 min. ExoQuick-TC™ (System Biosciences, USA) reagent was added to the media, mixed thoroughly, and incubated overnight at 4 °C to precipitate the EVs. The following morning, the mixture was subjected to centrifugation at 1500 g for 30 min, and the EV pellet was washed and resuspended in filtered PBS. The resuspended EVs were passed through the Exo-spin^™ columns (Cell Guidance Systems, St. Louis, MO, USA), and 300 μL of purified EVs were eluted from the column and used for experimental procedures. Protein concentration was determined using a protein assay kit from Bio-Rad, USA. The purified EVs were further characterized using nanoparticle size tracking and the determination of protein markers by Western blot analysis (Figure 1).