Amicon ultra 3k device

The TiO₂-NP suspension (rutile: anatase = 20: 80; secondary particle diameter <150 nm; primary particle diameter of starting nanopowder, 21 nm; Cat. No. 700347-25 G, Sigma-Aldrich Co. St. Louis, MO, United States) was passed through a 0.45 μm filter (Millex®-HV; Cat. No. SLHU033RS; Merck Millipore Ltd., Burlington, MA, United States) to eliminate aggregated particles and then diluted to 0.25, 1, and 4 μg/μL with distilled water. The particles in the filtered suspension were characterized by transmission electron microscopy (TEM; JEM 1200EXII, JEOL Ltd., Akishima, Tokyo, Japan) on collodion-coated 200 Cu mesh (Cat. No. 6511; Nisshin EM, Tokyo, Japan). The size distribution of secondary TiO₂-NP in the diluted suspension was determined by dynamic light scattering (DLS) measurements, using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, United Kingdom). For sham-treatment, a vehicle solution without any particles was prepared by centrifugation (18000 × g for 10 min at 4°C) of the TiO₂-NP suspension, followed by passing through an Amicon® Ultra (3 K) device (Merck Millipore). In the DLS analysis, no particulate signal was detected in the vehicle solution.

Pre-treatment of protein A/G magnetic beads (B23202, Bimake, China) was performed according to the manufacturer’s instructions. Then, the serum sample was diluted two-fold with PBS and was added to protein A/G beads (100 µl beads for 150 µl serum) to pre-capture the natural IgG in the serum (2 h-incubation with mixing at 4 °C). After the beads were removed, the sample was further diluted four-fold with PBS to reduce the concentration of non-target protein. The ChIP-grade anti-histone H3 antibody was added to the diluted sample (1:200) and incubated with mixing at 4 °C overnight. The antibodies were captured using pretreated protein A/G magnetic beads (150 µl beads for 800 µl sample) and the beads were washed with 200 µl PBS 3 times to eliminate non-specific binding. The remaining serum sample after bead-removal and 3 × 200 µl PBS bead-washing buffer were combined and concentrated using 3 K ultrafiltration tubes (Amicon® Ultra 3Kdevice, Millipore-Merck, U.S.A). cfDNA extracted from the concentrated sample was labeled as “cfDNA after pull-down”. cfDNA extracted from the magnetic beads was labeled as “cfDNA on beads”. Both fractions of cfDNA were used for subsequent concentration and size measurement.

For serum metabolite extraction, 50 µL of serum samples were mixed on ice with 120 μL methanol containing internal control substances: 25 μM 2-morpholinoethanesulfonic acid and 100 μM methionine sulfone. After centrifugation (16,000 × g for 10 min at 4 °C), 130 μL of supernatant was mixed with 250 μL ultrapure water and subjected to ultrafiltration using 3 kDa cutoff filters (Amicon Ultra 3 K device, Merck, Darmstadt, Germany), followed by 30 min of evaporation and 6 h of lyophilization.
For liver metabolite extraction, approximately 100 mg of liver pieces were homogenized in 500 μL of methanol containing internal controls. The homogenates were diluted with 250 μL of ultrapure water, and 600 μL of each homogenate was mixed with 400 μL chloroform. Following centrifugation (16,000 × g for 5 min at 4 °C), 800 μL of the upper phase was collected and subjected to 30 min of evaporation. Then, 300 μL of ultrapure water was added, followed by ultrafiltration and lyophilization.
The lyophilized metabolite samples were reconstituted in 200 μL ultrapure water, further diluted if necessary, and then subjected to LC–MS/MS (LCMS-8030, Shimadzu, Kyoto, Japan) analysis, using Method Package for Primary Metabolites ver. 2 (Shimadzu) according to the manufacturer’s protocol.

Six 5-week-old F1 juveniles (2 wild-type, 2 heterozygotes, and 2 sa9430 mutants) were homogenized by bead beating for 30 s at 4 m/s in 300 μL cold PBS with Pierce protease inhibitors (Thermofisher Scientific, Waltham, MA, USA). Samples were centrifuged for 5 min at 1500× g at 4 °C. Supernatant was filtered through an Amicon Ultra 3K Device (EMD Millipore, Billerica, MA, USA), dried, and then analyzed by HPLC as described in Section 4.8.

CKR12-PLGA-MCZ micelles were prepared using a solubilisation method. Briefly, a solution of MCZ (0.98 mg) in acetonitrile (100 μL) was added to a solution of CKR12-PLGA (500 μL) and sonicated for 1 min. The resulting emulsion was stirred overnight to completely remove acetonitrile at room temperature.
The total amount of MCZ entrapped in CKR12-PLGA-MCZ micelles was determined using an ultrafiltration method [35 (link)]. Briefly, the micelle solution was filled into an Amicon Ultra 3 K device (Merck, Kenilworth, NJ, USA) with a molecular weight cut-off of 3 kDa and centrifuged at 14,000× g for 5 min at 25 °C. The amount of MCZ encapsulated in CKR12-PLGA was calculated by measuring MCZ dissolved in the filtrate by HPLC [36 (link)].

The dialyzed sample was loaded on Hi Trap DEAE FF (1 ml, 7 × 25 mm) column (GE Healthcare), pre-equilibrated with 50 mM Tris–HCl buffer, pH 8.0. Akta purifier (GE Healthcare) connected system to control the flow rate and fraction size of elution. The sample was injected into the column with 5 ml loop. The flow rate of 1 ml/min is maintained for both binding and elution. The column was washed with equilibration buffer, and the bound proteins were eluted with NaCl linear gradient (0–1 M NaCl w/v) in the same buffer. The first peak obtained at 0.10 M of NaCl was pooled, concentrated using Amicon Ultra 3 K device (Merck Darmstadt, Germany).