Optima xl 100k

Both cell culture media and plasma samples were first triple-spun at 1600xg for 10 min. followed by two 3000xg spins for 10 min. to remove cellular debris. Samples were then processed via a 120,000xg spin for 2 hours at 4 degrees C per [44 (link)] in a Beckman-Coulter Optima XL-100K ultracentrifuge using a Beckman-Coulter SW28 rotor at the Extracellular Vesicle Core at the University of Pennsylvania.

The details of leaf TSP extraction were described in He et al. (2017a) (link). The frozen samples (1 g) were powdered in liquid nitrogen and ground with 6 ml of extraction buffer [100 mM Bicine-KOH (pH 8.1), 20 mM MgCl2, 2% PVP]. The mixture was centrifuged at 35,000 rpm for 30 min at 4°C using a Beckman ultracentrifuge Optima XL-100K. 1 ml aliquot of the supernatant was mixed with 4 ml of 80% cold acetone before centrifuging at 3500 rpm for 10 min. The precipitate was dissolved in 1 ml of 1 M NaOH. The concentration of leaf TSP was determined according to Lowry et al. (1951) (link).

A total of 10 g of feces was inoculated into 40 mL of sterile phosphate-buffered saline (PBS) and homogenized. Next, EVs were then isolated by centrifugation as previously described, with some modifications [8 (link),13 (link),24 (link)]. A first centrifugation of the homogenate was performed (40 min, 4000× g, 4 °C), and the supernatant was recovered and filtered using sterilized vacuum filtration units, Rapid-Flow™ filters MF 75, and Nalgene^® 0.2 μm in cold ice (ThermoFisher Scientific, Waltham, MA, USA). The filtrate was transferred to 10 mL polycarbonate open top thick wall tubes and ultracentrifuged at 100,000× g for 3 h at 4 °C, with a fixed angle rotor Type 70.1 Ti in a Beckman Optima XL-100K ultracentrifuge (Beckman Coulter Life Sciences, Lakeview Pkwy S Drive, Indianapolis, IN, USA). Pellets were resuspended in 200 µL of PBS and the EVs were purified using qEVoriginal size exclusion columns (SEC) of 70 nm (Izon Science Europe Ltd., Oxford, UK) [25 (link),26 (link)], following the manufacturer’s recommendations. Fractions 7–10 were collected, mixed, concentrated with Vivaspin^® 20 100 kDa (Sartorius Stedim Biotech GmbH, Göttingen, Germany) centrifugal concentrators, aliquoted and frozen at −80 °C until used.

S1 was centrifuged at 20,000×g at 4 °C in a Beckman Coulter ultracentrifuge Optima™ XL-100 K for 30 min using a type 70.1Ti rotor. The resulting pellet was discarded and the supernatant (S2) was centrifuged at 100,000×g in the same conditions for 1 h. The new supernatant (S3) was discarded and the resulting pellet containing the microsomal fraction (MP) was conserved for further LLO purification steps.

LDL particles were isolated from normal pooled human serum (Innovative Research, Novi, MI) using an established ultracentrifugation protocol [19 (link)]. Briefly, three different density solutions (1.006 g/mL, 1.02 g/mL and 1.065 g/mL) of KBR were prepared to enable the separation of the two layers of LDL (LF- and SD-LDL), which fall at the junction between each layer. The visualization (Fig 1) of the LDL heterogeneity was facilitated by prestaining the serum with Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, Waltham, MA) prior to density gradient ultracentrifugation using the Beckman Optima XL-100K. The centrifugation was done for 19.5 hrs at 30,600 rpm in 4°C.