Amicon ultra 15 100 kda centrifugal filter units

For lentiviral packaging, HEK293T cells were seeded in a 10 cm dish 1 day before transfection. The indicated viral plasmids Lentidcas9-vp64-blast (Addgene #61425) or FgH1tUTG (Addgene #70183) were co-transfected with lentivirus packaging plasmids pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) in a 4:2:3 ratio using PEI MAX (molecular weight 40,000) (Polysciences, Inc., USA). The next day after transfection, media were changed to Opti-MEM® I Reduced Serum Media (Thermo Fisher Scientific). At 48 h after transfection, virus-containing media were collected by Amicon Ultra-15 Centrifugal Filter Units 100 kDa (Millipore), passed through a 0.45 µm polyethersulfone filter (Millipore), aliquoted and stored at −80 °C before infection or titration. mESCs were seeded 1 day before transduction. The next day, the medium was changed to fresh and protamine sulfate 50 μg/ml (Sigma) and Lentidcas9-vp64-blast or FgH1tUTG were added. Infected green fluorescent protein-positive cells mESCs were sorted in BD FACSAria™ III. FgH1tUTG-transduced cells were infected with Lentidcas9-vp64-blast and selected by 5 µg/ml of blasticidin (Sigma) for 1 week. Oligonucleotides were designed using the online tools http://sam.genome-engineering.org/database and https://www.nature.com/articles/nbt.2647: F-TCCCGAGGCGGGGCGGGGCTTAGTT; R-AAACAACTAAGCCCCGCCCCGCCTC; and cloned into FgH1tUTG vector.

EVs were purified by differential centrifugation (Beckman TLA100.2 rotor) from the indicated conditioned media of monolayer cell cultures. After cell debris was eliminated by centrifugation at 2000 × g for 20 min, the supernatant was concentrated (centrifuged at 3500 × g for 20 min) using Amicon Ultra-15 Centrifugal Filter Units −100 kDa- (Millipore # UFC905008) to a final volume of 1 mL. The concentrated conditioned medium was passed through 0.22 μm filter and then centrifuged at 110,000 × g for 70 min. The resulting EV pellet was re-suspended in filtered 1 × PBS or RIPA buffer and stored at −80 °C.

EVs were purified by differential centrifugation (Beckman TLA100.2 rotor) from the indicated conditioned medium of monolayer cell cultures [31]. GSC were grown in their media, while differentiated cells were maintained in EV-depleted FBS. After three days, cell debris was eliminated by centrifugation at 2,000 × g for 20 min. The supernatant was then concentrated (centrifuged at 3,500 × g for 20 min) using Amicon Ultra-15 Centrifugal Filter Units −100KDa- (Millipore # UFC905008) to a final volume of 1mL. Concentrated conditioned medium was then centrifuged at 10,000 × g for 45 min to precipitate the 10K pellet (ectosome/microvesicle-like EVs). For isolation of exosome-like EVs, the supernatant remaining after the first centrifugation was passed through 0.22 μm filter and then centrifuged at 110,000 × g for 70 min. The resulting EV pellet was re-suspended in filtered 1× PBS or RIPA buffer and stored at −80°C (designated for simplicity as 100K).

EVs were purified as previously described^{3 (link)}. Briefly, the respective conditioned media were cleared of cells at 300 × g for 5 min, and dead cells/debris was further eliminated by centrifugation at 2000 × g for 20 min, after which the supernatant was concentrated by spinning at 3500 × g for 20 min using Amicon Ultra-15 Centrifugal Filter Units −100 KDa- (UFC905008, Millipore) to a final volume of 1 ml. The 1 ml Concentrate was subjected to ultracentrifugation at 110,000 × g for 1 h using a tabletop ultracentrifuge (Beckman TLA100.2 rotor). The pelleted EVs were washed with sterile PBS (311-425-CL, Wisent) at 110,000 × g for 1 h in the ultracentrifuge suspended in sterile PBS or RIPA buffer and analysed.