Exosome free fbs

About 2 × 10⁶ donor cells were plated in a culture dish with a diameter of 10 cm. Twenty-four hours later, the culture medium was replaced with serum-free DMEM and incubated for 48 h. For the CM from cisplatin-treated cells, the cells were incubated with serum-free DMEM containing 10 μM cisplatin. The donor medium was spun down at 3000×g for 10 min and stored at 4 °C. For long-term treatment of cells, the prepared CM was supplemented with 2% exosome-free FBS (SBI, USA). To obtain exosome-free CM, the CM was spun down successively at 300×g for 20 min, 2000×g for 20 min, and 12,000×g for 70 min to deplete exosomes from the media.

1×10⁶ GSC664 and GSC712 cells were cultured under normoxia or hypoxia for 48h, the culture medium was collected and supplemented with 2% exosome-free FBS (SBI, USA). 1×10⁶ TCs were cultured using this medium.

Passage 4 MSCs were expanded to 90% confluency (as described above) in complete growth medium containing exosome-free FBS (SBI, Palo Alto, CA, USA). Exosome-free medium was used to ensure that no contaminating exosomes were present from bovine serum. Conditioned medium (CM, 10 ml) was obtained after 48 h from confluent cultures and kept frozen at − 80 °C. Exosome extraction of CM was performed by SBI Biosciences using ExoQuick-TC reagent and miRNA extraction using SeraMir kit (SBI, Palo Alto, CA, USA). Quality and quantity of obtained RNA was verified using Agilent Bioanalyzer Small RNA Kit prior to generation of libraries using NEBNext Multiplex Small RNA Kit (New England Biolabs, Ipswich, MA, USA). Next generation sequencing was performed using Illumina HiSeq 2500 platform with 100 bp paired end runs (Illumina, San Diego, CA, USA). Sequencing data for all samples was normalized to picograms of miR.

Maintenance of CAD (catecholaminergic derived CNS cells) was described before^{56 (link)}. For expression in CAD cells, cDNAs encoding for CSPα and CSPα mutants were expressed in the plasmid myc-pCMV, HA-pCMV, pcDNA3.1-FLAG and pcDNA3.1-GFP. All mutations were confirmed by DNA sequence analysis. Constructs encoding Myc-tagged CSPα, Flag-tagged CSPα, HA-tagged Myc-tagged CSPα, Myc-tagged CSPα_L115R, Myc-tagged CSPα_ΔL116, Myc-tagged CSPα_HPD-AAA, GFP-72Q huntingtin^exon1 and SOD-1^G93A-FLAG were transiently transfected with Lipofectamine 3000 (Invitrogen) in Opti-MEM™ medium (Invitrogen). Media was changed to serum-free media 6 hrs post-transfection, or where indicated 5% exosome free FBS (Systems Biosciences). Where indicated, 50 µM resveratrol treatment (Sigma) started 6 hrs post-transfection.

For exosomes isolation, supernatant collected from ovarian cancer cells cultured in DMEM containing 10% Exosome-free FBS (System Biosciences, USA) for 48 h was centrifuged at 300 × g for 10 min, 3000 × g for 30 min and 10,000 × g for 30 min. Then, the supernatant was passed through 0.22-μm pore PES filters (Millipore). Ultracentrifugation was performed at 100,000 × g for 70 min using an Optima XE-90 Supercentrifuge (Beckman, Germany) to enrich the exosomes, and the pellet was rinsed using 30 ml of PBS. Finally, the exosomes were collected by ultracentrifugation at 100,000 × g for 70 min.
Isolated exosomes were mixed with 4% paraformaldehyde. exosomes were then dropped onto formvar carbon-coated electron microscopy grids and fixed with 1% glutaraldehyde for 10 min. Samples were negatively stained with 2% uranyl acetate solution. Images were obtained using Philips CM120 BioTwin transmission electron microscope (TEM) (FEI Company, USA).
Nanoparticle tracking analysis (NTA) was performed by Malvern Zetasizer Nano ZS-90 (Malvern Instruments Ltd., UK) following the manufacturer’s instructions. The exosomes were diluted in PBS. The mean particle size and size distribution were analyzed by dynamic light scattering method using the Malvern Zetasizer Nano ZS-90.