Optima tlx ultracentrifuge

The NCs production yield was determined by gravimetry. For that, fixed volumes of NCs suspensions were centrifuged (60,000 rpm, 1 h, 15 °C) in an Ultracentrifuge Beckman Coulter (Optima^TM TLX Ultracentrifuge. Rotor: TLA_100.3, Ramsey, MN, USA). The NCs creams were freeze-dried (over 24 h at −80 °C and gradual ascent until 20 °C), using a Telstar Freeze Dryer (Telstar LyoQuest −85, Barcelona, Spain) (n = 3).
The production yield (P.Y.) was calculated using the following formula: P.Y. % = NCs weightTotal solids weight× 100
where total solids = CS + (HA) + Lecithin + Miglyol^®

Vybrant DiD cell-labeling dye (Molecular Probes) was used for exosome labeling. Initially, 300 μg of freshly isolated exosomes was resuspended in 1 mL of PBS, and then, 5 μL of the DiD-dye was added according to the manufacturer’s instructions. Exosomes with dye were incubated for 15 min at 37 °C; then, 1 mL of 1% BSA in PBS was added, mixed, and kept for 2 min at 37 °C and centrifuged at 32,500 rpm (100,000×g) for 70 min at 4 °C (Optima^TM TLX Ultracentrifuge, Beckman Coulter). The DiD-tagged exosome pellet was re-suspended in 80 μL of PBS. The concentration of exosomes was measured by Nanodrop (Thermo Fisher Scientific), aliquoted with a small volume of 20 μL, and kept at -80°C until used.

Cells transfected with 4 μg of NiV N-CMV or NiV P-NiV N-CMV DNA were washed three times with sterile phosphate buffered saline (PBS) and lysed in PBS/0.1% Nonidet P40 (NP-40, Sigma-Aldrich, Oakville, Ontario, Canada) lysis buffer for 1 hour at 4 °C with end-over-end rotation. The cell lysate was clarified by centrifugation at 19,000 × g for 15 minutes and the supernatant was layered over 1.3 ml of a 20% sucrose cushion. The cushion was spun at 130,000 × g for 1 hour at 4 °C in a Beckman Optima^TM TLX Ultracentrifuge. The supernatant (soluble fraction) was collected and the pellet (insoluble fraction) was resuspended in an equal volume of sterile PBS. Sample analysis was done by western blot quantitation as previously described.

Type B toxic oligomers of α-synuclein were prepared as previously described^{33 (link)}. Briefly, the protein was purified into phosphate-buffered saline (PBS) and subsequently dialyzed against water (4 L; overnight, 4 °C). In all, 6 mg aliquots were lyophilized for 2 days, followed by resuspension in buffer (500 μL of 20 mM Tris, 100 mM NaCl, pH 7.4). The resuspended protein was passed through 0.22 μm filters and incubated (20–24 h, 37 °C). The samples were ultracentrifuged (1 h, 90,000 rpm, 20 °C) in a TLA120.2 rotor using an Optima TLX Ultracentrifuge (both Beckman Coulter, High Wycombe, UK) to remove aggregates and large oligomers. Any remaining monomeric protein was removed using a 100-kDa centrifugation filter (4×; 2 min, 10,000 rpm). The flow through containing predominantly monomeric protein from the first three passes was kept and reused up to five times. The oligomer concentration was determined by UV spectroscopy using an extinction coefficient of 5600 M⁻¹cm⁻¹ at 275 nm.

Cell fractionation assay protocol was modified from previous reports (Chua and Wong, 2013 (link)). Cells were harvested by trypsinization and washed with PBS. Then, cells were resuspended in 400 μl homogenizing buffer (8.6% saccharose, 25 mM HEPES pH 7.4, 5 mm MgCl, 1 mM EDTA, protease inhibitor 1x). Cells were lysed using 10 passes through a 22-gauge needle followed by 15 min incubation on ice. The homogenate was clarified by a 400 × g centrifugation for 5 min. The clarified homogenate was centrifuged at 100,000 × g for 2 h at 4°C (Optima TLX Ultracentrifuge, Beckmann Coulter). The cytosol fraction (supernatant) was separated from the membrane fraction (pellet) before the latter was resuspended with lysis buffer supplemented with protease inhibitors 1x. Both fractions were sonicated three times in a 5-s pulse at 10 Amp on ice. Cytosol and membrane fractions were incubated on ice for 1 h before a final centrifugation of 16,000 × g for 15 min. Clarified supernatants were subjected to protein quantification before WB analysis.

To prepare yeast cell lysates, cells were lysed using a CryoMill (Retsch) as described above and the cell powder was resuspended in 10% glycerol buffer (20 mM Tris pH 7.5, 137 mM NaCl, 10% Glycerol, 0.5% NP40, 2 mM EDTA). For human HepG2 cells, nuclear extract was prepared as described above and the nuclei were lysed in 10% glycerol buffer by passing through a 20G needle 10 times. The lysate was resolved by loading 150 µg onto a linear 20–50% glycerol gradient prepared in an ultracentrifuge tube (Beckman, 347357). The gradients were spun in an Optima TLX-ultracentrifuge (Beckman) at 81,400 × g for 19 h. Fractions were collected by pipetting and were resolved on a 4–12% gradient gel (Invitrogen, NP0336BOX), followed by western blot analysis with ARS2 and EFTUD2 antibodies as described above. Western blotting to detect S. pombe proteins was performed with the following antibodies: GFP (Pir2-GFP detection; Roche, 11814460001) at dilution 1/1000, HA (Cdc5-HA and Spp42-HA detection; Biolegend, 901501) at dilution 1/1000 and FLAG (Cbc1 detection; M2 Sigma, F1804) at dilution 1/1000.