Ultra clear

Late endosomes were isolated using sucrose gradients as described previously [43 (link), 54 (link)]. Briefly, 25 × 10⁶ CHO-WT and CHO-A6 cells were used for each gradient. Cells were washed twice with cold PBS and collected. Cells were pelleted and resuspended in homogenization buffer (250 mM sucrose, 3 mM imidazole, pH 7.4) supplemented with protease/phosphatase inhibitors cocktail (see above). Next, cells were homogenized by 15–20 passages through a 22 G needle at 4 °C. Complete homogenization was confirmed under the phase microscope. The homogenate was centrifuged for 15 min at 1000g at 4 °C. The post-nuclear supernatant was collected and quantified by Bradford and 3 mg of PNS were brought to a final 40.2% sucrose (w/v) concentration by adding 2.5 M sucrose and loaded at the bottom of a 13.2-ml tube (Beckman UltraClear). Then 3 ml of 35% sucrose, 3 ml of 25% sucrose and 2.5 ml of homogenization buffer were overlaid stepwise on top. The gradient was centrifuged for 90 min at 150,000g, 4 °C in a Beckman SW 41 Ti rotor. After centrifugation, 1.5-ml fractions were collected from top to bottom and protein was precipitated using trichloroacetic acid/acetone to determine the TBC1D15 and Rab7 distribution by western blotting.

All conditioned bacterial culture media was centrifuged two times at 7,000 g for 15 min at 4 °C and then 0.45 μm membrane filtered. This removed all intact bacteria and undesired large cellular material. OMVs (36 mL of culture media) were then pelleted in Ultra-Clear (25 × 89 mm) centrifuge tubes purchased from Beckman Coulter (Brea, CA) at 29,000 RPM (~150,000 g) in a Sorvall WX Ultra 90 centrifuge using an AH-629 rotor for 3 h at 4 °C. The OMV depleted culture media was decanted and the OMV pellet was resuspended in 1 mL of PBS pH 7.4.

Lipid raft and non-raft fractions were isolated following the protocol described in Fabelo et al. (2012 (link), 2014 (link)). Briefly, 0.1 g of cerebellar or occipital cortices were homogenized at 4°C in 8 volumes of isolation buffer (IB: 50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 0.15 M NaCl) containing 1% Triton X-100 and 5% glycerol, 20 mM NaF, 1 mM Na₃VO₄, 5 mM β-mercaptoethanol, 1 mM PMSF, and a cocktail of protease inhibitors (Roche Diagnostics, Barcelona, Spain) in a glass homogenizer for 5 min, centrifuged at 500 g for another 5 min, and then the supernatant was collected and mixed in an orbital rotor for 1 h at 4°C. About 800 μL of the supernatant was mixed with an equal volume of 80% sucrose in IB and overlaid with 7.5 mL of a 36% sucrose solution and 2.7 mL of a 15% sucrose solution in IB, in 10 mL ultracentrifuge tubes (Ultraclear, Beckman). Sucrose gradients were centrifuged at 150,000g for 18h at 4°C using a Beckman SW41Ti rotor. Two mL fractions were collected from the top to the bottom. Fractions 1 and 2, which contained the lipid raft resident protein markers, were pooled together and identified as lipid raft fractions. Non-raft fractions corresponded to fraction 6 and the pellet which contained the non-raft resident protein markers. Fractions were frozen at -80°C until analysis.

EVs were collected when cells reached approximately 80% (ASC) or 90% (HUVEC, hiPSC-ECFC) confluency. Full medium was removed and the cells were washed 3× with 1 × PBS (w/o Ca⁺⁺ Mg⁺⁺) to eliminate any contaminating EVs from the FCS. Endothelial Basal Medium (EBM-2, Lonza, Walkersville, MD, USA) was applied and conditioned for 48 h. Two identical flasks of each cell type were cultured using one for EV collection and one to determine cell number prior to conditioning. As a control EBM-2 w/o cells was used. The conditioned medium from each cell type was centrifuged at 500× g for 5 min at 4 °C in a fixed angle rotor (SN867, Heraeus Megafuge 16R). The resulting supernatant, termed “S0.5”, was centrifuged again at 2000× g for 5 min at 4 °C to remove cell debris. The supernatant (“S2”) was transferred into ultracentrifugation tubes (Ultra-Clear, Beckmann Coulter, CA, USA) and centrifuged at 100,000× g for 65 min (including acceleration time) at 4 °C using a swing out rotor (SW40.1 Ti) in the ultracentrifuge L-100XP from Beckman Coulter. The obtained pellet “P100” containing small and large EVs was resuspended in 1 mL sterile-filtered (0.22 µm PVDF filter) 1 × PBS (w/o Ca⁺⁺ Mg⁺⁺). “P100” and an aliquot of supernatant “S100” were stored at 4 °C until further use.