L8 m ultracentrifuge

ES cells, grown to 80% confluence, were incubated with 1% (vol/vol) of 9 mg/mL cycloheximide (Sigma Aldrich) for 10 minutes at 37°C and then trypsinized. Cells were washed with PBS and lysed at 4°C using a handheld homogenizer (Fisher Scientific) in polysome buffer containing 50 mM Tris-HCl (Fisher Scientific), 240 mM NaCl (Fisher Scientific), 10 mM MgCl₂ (Sigma Aldrich), 5 mM beta-mercaptoethanol (Sigma Aldrich), 250 mM sucrose (Fisher Scientific), 2% Triton X (Sigma Aldrich), 100µg/mL heparin (Alfa Aesar), and 90µg/mL cycloheximide. Lysates were run on 15–55% sucrose gradients containing 25 mM Tris-HCl, 25 mM NaCl and 5 mM MgCl₂. Gradients were centrifuged at 28,000 rpm for 7–8 hours using a Beckman L8-M ultracentrifuge. The gradients were then broken down using an ISCO density gradient fractionator, retriever and UA-6 UV/Vis detector (ISCO).

The cusS gene and its derivatives were cloned into pASK-IBA7 (IBA, Göttingen, Germany), which attaches an amino-terminal Strep Tag II under the control of the tet promoter in E. coli strain BL21(pLys). Precultures in LB were diluted 50-fold into fresh LB and were incubated with shaking at 37°C until the turbidity at 600 nm reached 1.0. Anhydrotetracycline (AHT) was added to a final concentration of 10 or 200 μg/L. Incubation was continued with shaking at 30°C, and the cells were harvested by centrifugation. Crude extract was prepared by ultrasonication followed by removal of the debris by centrifugation for 30 min at 4°C and 5,000 rpm (Eppendorf 5804R, Eppendorf, Hamburg). The crude extract was further separated into membrane and soluble fractions by ultracentrifugation (1 h at 45,000 rpm and 4°C; Beckman L8-M ultracentrifuge). Following SDS-PAGE (52 (link)) of samples representing 50 μg of dry mass or 25 μg of protein in a linear 10% (wt/vol) gel, the proteins were transferred by semidry electroblotting onto a nitrocellulose membrane (NCP, Macherey-Nagel, Düren, Germany) at 3 mA/cm² and 15 V for 30 min and detected using a Strep-Tactin peroxidase conjugate antibody (Institut für Bioanalytik Göttingen, Germany) and chemiluminescence detection, as described by the manufacturer (Lumi-Film, Roche, Mannheim, Germany).

After 48 h incubation, the conditioned media was collected and centrifuged at 300xg for 10 min to pellet cells. The supernatant was collected and spun at 2,000xg for 20 min to remove dead cells, followed by storage at -80 °C until EV isolation.
To isolate the EVs, the conditioned medium underwent differential ultracentrifugation using a 30% sucrose/deuterium oxide (Sigma-Aldrich, Poole, UK) cushion, made up to a density of 1.210 g/cm³ [22 ]. The conditioned media was passed through a 0.22 µm filter (Sarstedt, Leicester, UK), loaded onto a 30% sucrose cushion, and then centrifuged at 100,000xg for 1 h 45 min on an SW28Ti rotor, 25PC Polycarbonate open-top tubes (Scientific Laboratory Supplies, Nottingham, UK) and using an L8-M Ultracentrifuge; k-factor 296.8 (Beckman Coulter, High Wycombe, UK). Using a 21G needle and syringe, 3-4 ml of EV suspension was collected at the interface between the sucrose cushion and conditioned media. DPBS was added to this suspension, and it was centrifuged on a Type 70.1Ti fixed angle rotor at 100,000xg for 60 min to pellet pure EVs. All ultracentrifugation experiments were performed at 4 °C and EV pellets were stored at -80 °C. For clarity, this paper uses the term ‘extracellular vesicles’ to describe both exosomes and microvesicles which are smaller than 0.22 µm and float at a density of < 1.2 g/ml, and hence represent small EVs.

Cells were isolated on ice by washing 2× with cold PBS and 1× with homogenization buffer (HB; 20 mM tris pH 7.4, 250 mM sucrose, 1 mM EDTA, protease inhibitor as needed). Cells were collected in HB + protease inhibitor and homogenized in Dounce homogenizer. A post-nuclear supernatant was carefully layered on top of a prepared 5%, 10%, 15%, 20%, 25% (top to bottom) OptiPrep gradient in a clear ultracentrifuge tube. Samples were spun at 40,000 rpm in a Beckman L8-M Ultracentrifuge for 3 h at 4 °C under vacuum in a SW41 swing rotor. Fractions were collected by aliquoting 800 μL from the top of the ultracentrifuge tube. Aliquots of each fraction were removed for protein determination with a modified Lowry assay. The samples were analyzed for protein by Western blot. For cholesterol analysis, the lipids were extracted with chloroform: methanol (2:1) and spotted on a TLC plate. Cholesterol was separated using the solvent system hexanes: ethyl ether: acetic acid (30:20:1). To visualize the lipids, plates were submerged briefly with 3% Copper acetate (w/v) in 8% phosphoric acid (v/v) and heated at 180 °C for approximately 15 min to char [103 (link)].