Optima l 100 xp ultracentrifuge

Medium-sized eels (length 0.7–1.5 m) were killed by decapitation after hypothermia. The electroplax organ was dissected, cut into 2-cm slices and stored in liquid nitrogen. Thawed electroplax tissue was homogenised for 30 s with an HR1610/01 hand blender (Philips) at full speed in 4 vol. of buffer A (0.05 M MOPS pH 6.5, 5 mM Na₂EDTA, complete protease inhibitors tablets). The homogenate was strained through a filter using a cafetiere jug (Sainsbury's), and 4 layers of gauze tissue. It was centrifuged in a Ti45 rotor in an Optima^TM L-100 XP Ultracentrifuge (Beckman Coulter, Inc.) at 36,000 × g for 30 min, at 4 °C. Pellets were pooled, homogenised using the hand blender (Philips) in 4 vol. of buffer A, and centrifuged again as before to give crude membranes. Membranes were homogenised in 0.5 vol. of buffer B (0.05 M MOPS, pH 6.5), and stored in liquid nitrogen.

Conditioned media were collected, and dead cells and debris were removed by centrifugation at 1500× g for 5 min. Supernatants were mixed with 99.9% (v/v) ice-cold molecular grade acetone at a ratio of 1:5 in a 50 mL centrifuge tubes and incubated overnight at −20 °C for protein precipitation. Samples were then subjected to initial centrifugation at 1700× g for 5 min (Haraeus Megafuge 1.0) and the supernatants were collected and further centrifuged at 12,000× g for 10 min at 4 °C to precipitate out the proteins (SW28 rotor in Beckman Coulter Optima^TM L-100XP Ultra centrifuge). For this purpose, thin-wall polypropylene centrifuge tubes (Beckman Coulter) were used. Supernatants were concentrated using a Speed vac concentrator (SAVANT SPD 131 DDA, Thermo Scientific, Waltham, MA, USA) to first remove the more volatile acetone. The protein depleted samples were then freeze dried (Martin Christ Beta 2–8 LD Plus) and stored at −80 °C until further processing and analysis.

The real concentration of REO in MLNs was determined using a direct method, as described below. In brief, a known amount of nanoparticles suspensions was first diluted (1:3) in water and then separated by gradient ultracentrifugation (271,000× g) at 4 °C for 1 h using Optima TM L-100XP Ultracentrifuge (Beckman Coulter Inc, Brea, CA, USA). The lipid supernatant was carefully removed, and 0.07 g of it was dissolved in 1.5 mL of chloroform (with 1% trans-2-hexenal as an internal standard) in an ultrasonic bath for 15 min. The obtained solution was further vortexed and filtered (PTFE, 0.22 µm), and the REO amount was analyzed by gas chromatography coupled with mass spectrometry (GC–MS), as detailed in Section 3.3.4.
The EE (%) and the LC of REO were calculated using the following equations: $$\mathrm{REO}\text{-}\mathrm{EE} (\%)=\frac{\mathrm{Real} \mathrm{concentration} \mathrm{of} \mathrm{encapsulated} \mathrm{REO}}{\mathrm{Theoretical} \mathrm{concentration}}\times 100$$
$$\mathrm{REO}\text{-}\mathrm{LC} (\frac{\mathrm{mg}}{\mathrm{mL}})=\frac{\mathrm{mass} \mathrm{of} \mathrm{encapsulated} \mathrm{REO}}{\mathrm{Total} \mathrm{volume} \mathrm{of} \mathrm{MLN}}$$

Exosomes were prepared from neuronal primary culture NCM. The medium was collected and centrifuged at 300×g for 10 min and 2000×g for 10 min at 4 °C. Following centrifugation, the supernatant was passed through a 0.22-μm sterile filter (Steritop™ Millipore, MA, USA). Then, the filtered supernatant was transferred to the upper compartment of an Amicon Ultra-15 Centrifuge Filter Unit (Millipore) and centrifuged at 4000×g until the volume of the upper compartment was reduced to ~ 200 μL. The liquid was loaded onto the top of a 30% sucrose/D2O cushion in a sterile Ultra-Clear™ tube (Beckman Coulter, CA, USA) and underwent a 10,000×g centrifugation step for 60 min at 4 °C in an Optima L-100 XP Ultracentrifuge (Beckman Coulter) in order to purify the exosomes. Partially purified neuron-derived exosomes were recovered using an 18-G needle diluted in PBS and centrifuged at 4000×g at 4 °C in a filter unit until the final volume reached 200 μL. Exosomes were used for downstream experiments or stored at − 80 °C
A Nanosight LM10 System (Nanosight Ltd., Navato, CA) was used to analyze the distribution of vesicle diameters from the exosomes. A transmission electron microscope (TEM; Tecnai 12; Philips, Best, The Netherlands) was used to observe the morphology of the acquired exosomes. Western blotting was used to determine specific exosome surface markers, such as CD9, CD63, and CD81.

A urine sample (60-100 mL) was dialyzed against ddH₂O overnight using 3.5kDa molecular weight cut-off SpectraPor membranes (Spectrum Labs, Rancho Dominguez CA). The next day, the sample was filtered through a Nalgene Rapid Flow filtration unit with a 0.2 µm aPES membrane (Thermo Scientific, Rockford IL), treated with 0.02% sodium azide, and Complete^® EDTA-free protease inhibitor (Thermo-Pierce, Rockford IL). The samples were then loaded into 29.9 mL Beckman Optiseal Tubes and spun at 100,000 × g on a 70 Ti fixed angle rotor in a Beckman Coulter Optima L100 XP Ultracentrifuge (Beckman Coulter, Indianapolis IN) for 90 minutes at 4°C. Samples were then resuspended in cell culture grade 1× PBS (Cellgro without calcium or magnesium Corning, Manassas VA) with protease inhibitor. Samples were stored at 4°C until use.

Crude OMVs were obtained by ultracentrifugation of the filtered spent medium from 150 mL of liquid culture as described (21 (link)). Briefly, 18- to 20-h cultures of B. thetaiotaomicron were centrifuged at 6,500 rpm at 4°C for 10 min. To remove residual cells, the supernatant was filtered using a 0.22-mm-pore membrane (Millipore). The filtrate was subjected to ultracentrifugation at 200,000 × g for 2 h (Optima L-100 XP ultracentrifuge; Beckman Coulter). The supernatant was discarded, and the pellets containing the OMV preparation were resuspended in phosphate-buffered saline (PBS) and normalized by OD before protein and lipid analyses. The OMV amount was estimated by measuring the protein content using a DC protein assay kit (Bio-Rad). Fractions were aliquoted and stored at −80°C until analyzed.