Amicon ultra 4 10 kda centrifugal filter

Extracellular vesicles (EVs) were isolated using the size exclusion column method as we described previously [16 ]. Briefly, the conditioned medium was collected, and EVs were isolated by centrifugation at 3000 rpm for 30 min to remove cells and debris, followed by filtration through a 0.22-μm filter to remove the remaining debris. Then, the medium was further concentrated using Amicon Ultra-15 100 kDa centrifugal filter units (Millipore). Isolation of EVs in the concentrated medium was carried out through the qEV size exclusion columns (Izon Science). EV fractions were collected and concentrated by Amicon Ultra-4 10 kDa centrifugal filter (Millipore). The purified EVs were stored at − 80 °C and subsequently characterized by particle size and electron microscopy.

Extracellular vesicles were isolated using the size exclusion column method. Briefly, conditioned media were collected, and EVs were isolated by centrifugation at 3000 r/min for 30 min to remove cells and debris, followed by filtration through a 0.22 μm filter to remove the remaining debris. Then the medium was further concentrated using Amicon Ultra-15 100 kDa centrifugal filter units (Millipore). Isolation of EVs in the concentrated medium was carried out through qEV size exclusion columns (Izon Science). EV fractions were collected and concentrated by an Amicon Ultra-4 10 kDa centrifugal filter (Millipore). The purified EVs were stored at -80°C and subsequently characterized by particle size, electron microscopy, and proteomic profile.

The expression vector was transformed into E. coli BL21 (DE3) cells (Novagen). For large scaled protein expression, cultures were grown in LB medium of 0.8 liter at 37°C for 4 h, induced with 0.4 mM isopropyl-β-_D-thiogalactopyranoside, and incubated overnight at 20°C. After centrifuging at 6,000 x g at 4°C for 15 min, the cell pellets were resuspended in lysis buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 5% glycerol, 0.2% Triton X-100, and 2 mM β-mercaptoethanol) and then lysed by sonication. The crude extract was then centrifuged at 12,000 x g at 4°C for 25 min to remove the insoluble pellet. The supernatant was incubated with 1-ml Ni-NTA beads at 4°C for 1 h and then loaded into an empty column. After allowing the supernatant to flow through, the beads were washed with washing buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 8 mM imidazole, and 2 mM β-mercaptoethanol), and the protein was eluted with elution buffer (20 mM Tris, pH 8.5, 30 mM NaCl, 150 mM imidazole, and 2 mM β-mercaptoethanol). The protein was then loaded onto a S-100 gel-filtration column (GE Healthcare) equilibrated with running buffer (20 mM Tris, pH 8.5, 100 mM NaCl, and 2 mM dithiothreitol). The purity of the fractions collected was analyzed by SDS-PAGE and the protein was concentrated to 30 mg/ml by Amicon Ultra-4 10-kDa centrifugal filter (Millipore).