Tla 110 fixed angle rotor

Density-gradient separation of DNA was performed according to Buckley et al. [23 (link)] with the exception of excluding the secondary bis-benzimide CsCl gradient [24 (link)]. In summary, DNA fragments >4 kb were selected using a BluePippin platform (Sage Science, Beverly, MA, USA) and added to gradient buffer (15 mM Tris-HCl, pH 8.0; 15 mM EDTA; 15 mM KCl) containing 1.762 g ml⁻¹ CsCl in 4.7 ml polypropylene tubes. Samples were ultracentrifuged to isopycnic equilibrium at 164 000 rcf for 66 h at 20 °C in a TLA-110 fixed angle rotor (Beckman Coulter, Brea, CA, USA). Following centrifugation, tubes were fractionated from bottom to top in 100 μl increments via displacement by Milli-Q water. Fraction densities were calculated using an AR200 Digital Refractometer (Reichert Technologies, Depew, NY, USA). Fractions were then desalted using an Ampure XP bead clean-up kit (Beckman Coulter) and stored at −80 °C.

The Wang and colleagues 2017 method, which includes a 0.22 μm filtration step (as previously described [25 (link)]), was used to remove most intact mitochondria from the EV preparation, which typically range between 0.2 and 1 μm. Briefly, supernatants from NSCs (cultured as above) were collected, transferred to 50 ml polypropylene centrifuge tubes, and centrifuged for 10 minutes at 300 x g. The supernatants were then collected and transferred into new 50-ml polypropylene centrifuge tubes before being subject to further centrifugation for 30 minutes at 2,000 x g. Subsequently, supernatants were transferred to 100-ml polycarbonate tubes and centrifuged for 20 minutes at 16,500 x g. Supernatants were transferred to new polycarbonate tubes prior to ultracentrifugation at 100,000 x g for 70 minutes at 4°C using an Optima XPN-80 ultracentrifuge with SW 32 Ti swinging rotor (Beckman Coulter). Pellets were immediately resuspended in ice-cold PBS and then filtered through a disposable filter unit (0.22 μm). Supernatants were pooled together in ultracentrifuge tubes and centrifuged for 30 minutes at 4°C at 100,000 x g using an Optima MAX ultracentrifuge with a TLA-110 fixed angle rotor (Beckman Coulter). Pellets resulting from this step, called EVs^Mito-depl., were used for subsequent applications.

Peripheral blood was collected at each visit in EDTA tubes and centrifuged at 1500 × g for 15 min to separate the serum fraction following standard operating procedure. Aliquots of cell-free serum were stored immediately at −80 °C and thawed just before use for this study. For the isolation of EVs and miRNA extraction we used the ultracentrifugation method as first described by Théry et al. [26 ] with some modifications [27 ]. In brief, serum samples (~1.5 mL) were thawed on ice and centrifuged at 1000, 2000, and 3000 x g for 15 min at 4 °C, consecutively, to remove any remaining cell debris and large aggregates. Supernatants were then filtered using a 0.8-μm membrane unit (Millipore Corp., Bedford, MA) and further centrifuged using a TLA-110 fixed-angle rotor (Beckman Coulter, Danvers, MA) at 110,000 x g (k factor of 13) for 2 h at 4 °C. Isolated EVs were visualized by transmission electron microscopy and immune-gold labeling using antibodies for the CD-63 and CD-81 surface markers as described by Théry et al. [26 ]. (Additional file 1: Figure S1). Finally, miRNAs were extracted from the collected EVs using the miRNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions, and the RNA eluate was concentrated for downstream analysis using a vacuum concentrator.