Amicon ultra 15 centrifugal filter device

EVs in CM were isolated using qEV10 / 35 nm size exclusion columns (IZON, Lyon, France) according to the manufacturer’s instructions. Briefly, cells, cell debris, and larger vesicles were removed by serial centrifugations at 300 × g at 4 °C for 10 min, 2000 × g at 4 °C for 10 min. CM samples were filtered through a 0.22 μm membrane. CM was concentrated using Amicon® Ultra-15 centrifugal filter devices (Merck, Stockholm, Sweden). Flush the qEV10 / 35 nm size exclusion columns with 90 ml PBS. Cell supernatant was loaded and the 20 ml void volume was immediately collected. The following 20 ml EVs samples after void-volume was collected. EVs samples were concentrated with Amicon® Ultra-15 centrifugal filter devices (Merck). Final EV preparations were stored in PBS supplemented with human albumin and trehalose (PBS-HAT) buffer at -80 °C until usage [58 ].

Escherichia coli (E. coli) BL21 (DE3; TaKaRa) carrying vectors expressing GST or GST‐fusion proteins were grown in LB (Sangon Biotech, Shanghai, China) at 37 °C with shaking until OD₆₀₀ was about 0.6, followed by addition of 0.1 mm IPTG (Sangon Biotech) and incubation at 22 °C for another 8 h. The E. coli were resuspended in ice‐cold PBS supplemented with protease inhibitor cocktail and 1 mm DTT, sonicated at 4 °C for 60 min (5 s on and 15 s off) on Bioruptor under the high‐power model, then centrifuged at 15 000 g and 4 °C for 30 min. GST‐fusion proteins in the supernatants were purified by GST‐sefinose (TM) resin (Sangon Biotech) according to the manufacturer's instructions. All proteins were concentrated in BC100 buffer (20 mm Tris–HCl at pH 8.0, 0.5 mm EDTA, 100 mm KCl, 20% glycerol, 0.5 mm DTT, 0.5 mm PMSF [Beyotime], and protease inhibitor cocktail) by Amicon Ultra‐15 Centrifugal Filter Devices (UFC901008; Millipore, Billerica, MA) and stored at −80 °C. The protein concentration was measured by BCA Protein Assay Kit (Beyotime).

Prophage were induced using a modification of the method of McShan et al. [54 (link)]. Briefly, WT SF370 (Sm^S), CEM1ΔΦ, and other phage cured mutants were grown overnight at 37°C in BHI broth. The overnight culture was diluted 1:30 (v/v) in 100 ml of BHI broth and incubated at 37°C to an O.D.₆₀₀ of 0.2. The culture was then divided, one half received mitomycin C (Sigma) at 0.2 μg/ml and the other half served as a control. After two hours at 37°C the optical density of the mitomycin C induced culture of SF370 began to drop. This decrease was not observed in either the untreated controls or the mitomycin C treated CEM1ΔΦ cultures (data not shown). Cells were then pelleted by centrifugation and supernatants collected and sterile-filtered through 0.22 μm filters. Filtrates were concentrated and buffer exchanged with 100,000 MWCO Amicon Ultra-15 centrifugal filter devices (Millipore) to achieve a final volume of 500 μl in prophage suspension buffer [55 (link)] (0.15 M NaCl, 10 mM Tris HCl [pH 7.5], 5 mM MgCl2, 1 mM CaCl2) These concentrated phage stocks were stored at 4°C for a maximum of 72 hours before being used.

The CDPH (1:10
diluted) was filtrated with Amicon Ultra-15 centrifugal filter devices
(Millipore) with a cutoff of 3 kDa and inserted in 50 mL centrifuge
tubes following the manufacturer’s instructions. The dialysis
of the whole CDPH or the filtrated CDPH was performed in distilled
water in the 20 mL Pur-A-Lyzer tubes (molecular mass cutoff 1 kDa,
Sigma Aldrich) as described by the manufacturer.
Fast protein
liquid chromatography (FPLC) fractionation of the CDPH was carried
out on an FPLC GE Healthcare AKTA pure (GE Healthcare) system. All
FPLC runs were performed at room temperature and the elution profile
was monitored observing the absorbance values at 214 nm. Size-exclusion
chromatography was carried out on a single Superdex 30 Increase 10/300
GL column (GE Healthcare) eluting 0.5 mL of the 1:100 CDPH in a phosphate-buffered
saline (PBS) degassed solution (20 mM Na₂HPO₄, 20 mM NaH₂PO₄, 150 mM NaCl, pH = 7.4) at
a 0.5 mL· min^–1 flow. Similarly, a run was
performed to obtain a reference spectrum co-eluting six different
molecular standards, that is, 0.05 mg·mL^–1 bovine
serum albumin (M_r 66 463), 0.2
mg·mL^–1 cytochrome C (M_r 12 400), 0.2 mg·mL^–1 aprotinin
(M_r 6500), 0.07 mg·mL^–1 vitamin B12 (M_r 1355), 0.2 mg·mL^–1 tryglycine (M_r 189),
and 14 mg·mL^–1 glycine (M_r 75). Prior to each sample injection into the system, one
column volume of eluent buffer was run to ensure equilibration of
the column.

The culture supernatant of HepG38.7-Tet cells^{14 (link)} was concentrated using Amicon Ultra-15 Centrifugal Filter Devices (MILLIPORE, Darmstadt, Germany) and the resulting HBV sample (HBV DNA copies 2 × 10⁸/ml) was used as an inoculum for infection assays. HepG2-hNTCP-C4 cells cultured in a 24-well collagen-coated plate were transfected with pcDNA-F-PUF60 and then inoculated with the HBV sample (50 µl) in DMEM containing 4% polyethylene glycol (PEG) 8000 (Promega) after 12 h of transfection. The cells were washed 3 times with PBS after 24 h of infection and then subjected to RT-qPCR after 96 h of further culture.

ADSCs at passage 3 were cultured for 48 h in DMEM containing 10% exosome-depleted FBS, which was obtained by ultracentrifugation at 120,000 × g for 18 h. The cell culture supernatant was collected by concentrating the conditioned medium in 100 K MWCO Amicon®Ultra15 Centrifugal Filter Devices (Millipore, USA). Exosomes were isolated using ExoQuick-TC (SBI Biosciences) at a ratio of 1 : 5 (ExoQuick-TC: supernatant) according to the manufacturer's instructions [18 (link)], resuspended in 200 μL PBS, and stored at −80°C for subsequent experiments. The exosome protein levels were quantified with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Exosomes were characterized by examining the particle size, morphology, and protein markers based on MISEV2018 minimal information proposed by the International Society for Extracellular Vesicles (ISEV) [19 (link)]. The size distribution of exosomes was analyzed by nanoparticle tracking analysis (NTA) using a ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany). The morphology of exosomes was observed by transmission electron microscopy (JEM-1230; JEOL, Japan). Markers of exosomes, including TSG101, CD9, and CD81, were verified by western blot analysis.