Amicon ultra 4 10 kda centrifugal filter device

The sEV eluate and sEV-depleted fraction were concentrated using an Amicon Ultra-4 10 kDa centrifugal filter device (Merck Millipore). The protein concentration was determined via the BCA assay, and 20 μg of protein was resolved on SDS-PAGE, transferred to the PVDF membrane, and blotted with primary antibodies specific for CD63 (10628D, Thermo Fisher Scientific, USA, 1:1000), transferrin (ZRB1225, Sigma-Aldrich, USA, 1:1000), Calnexin (Adi-SPA-860-D, Enzo Life Sciences, Farmingdale, USA, 1:1000) and GM130 (610822, BD Transduction Laboratories, USA, 1:1000). Antibody labeling was detected by incubating blots with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (BR1706516, Bio-rad, USA, 1:4000) and PierceTM ECS Plus Western Blotting Substrate and captured with Amersham Imager 600 (GE Healthcare Life Sciences).

Exosomes were isolated from the pooled serum samples of each group by size exclusion chromatography and the Total Exosome Isolation Reagent (Thermo Scientific) according to the manufacturer’s instructions. For size exclusion chromatography, qEV columns (Izon Science) were rinsed with phosphate-buffered saline (PBS) before adding the pooled serum samples (500 μl) and fractions (0.5 ml) were collected. Three EV-rich fractions (7 (link)–9 (link)) were pooled and concentrated using an Amicon Ultra-4 10 kDa centrifugal filter device (Merck Millipore). We also isolated EVs using the Total Exosome Isolation Reagent kit. The required volume of pooled serum sample was first diluted with an equal volume of PBS to decrease viscosity before adding 0.2 volumes of the Total Exosome Isolation Reagent. The mixture was vortexed and incubated at 4°C for 30 min. The sample was then centrifuged at 10,000 ×g for 10 min at room temperature and the pellet from every 100 μl serum was resuspended in 25 μl PBS for Western blotting and Transwell assays. A fraction of the resuspended isolated exosomes were lysed with RIPA buffer and the protein concentration was measured using a BCA protein assay kit (Thermo Scientific).

Approximately 10 mL peripheral blood was collected with EDTA tube, centrifugated at 4 °C, 3000× g, 10 min for separating plasma and blood cells, then centrifugated at 25 °C, 1800× g, 8 min for obtaining lymphocytes after lysis red blood cells. Extracted blood components were stored at −80 °C for future experiments. EV was isolated from plasma samples by using the SEC-based qEV column (Izon Science, Christchurch, New Zealand), and the centrifugation method (CP100NX, Hitachi, Tokyo, Japan) was also applied in this work for EVs extracting. Finally, EV eluates were filtrated with an Amicon Ultra-4 10 kDa centrifugal filter device (Merck Millipore, Burlington, MA, USA).