Sorvall wx floor ultra centrifuge

iPSC-MSCs were pre-incubated with 5 μg/mL Dxl (Sigma, PHR1883) for 24 h or not pretreated, and then broken down by serial extrusion in the presence of 50, 100, or 200 μg/mL Dxl to make Dxl-loaded NVs. The amount of Dxl loaded into NVs was determined by UV spectrometry at 230 nm as reported [25 (link)] using a spectrofluorometer (ThermoFisher). After incubation in 37 °C PBS containing 10% pooled human serum (Sigma) or 4 °C PBS for a series of periods, the supernatant was isolated by ultra-centrifugation at 100,000×g for 90 min at 4 °C using Sorvall WX Floor Ultra Centrifuge (Thermo) to measure Dxl release from NV-Dxl by UV spectrometry. Dxl-resistant PC3 cells were established as reported [26 (link)]. 5 × 10³/well parent PC3 cells, Dxl-resistant PC3 cells, or THP-1 human myeloid cells were seeded into 96-well plates, incubated with empty NVs, NV-Dxl, or free Dxl at a series of concentrations for 72 h, and then analyzed with PrestoBlue Cell Viability Assay (ThermoFisher).

Membrane and cytosol fractions were isolated from kidney tissue of P2x6^-/- and P2x6^+/+ littermates by ultracentrifugation. To this end, half of a mouse kidney was first homogenized using a homogenizer (bedrijf) in 1 ml of lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM NaOrthovandate, 5 mM NaF, 5 mM glycerolphosphate 0.27 M sucrose) containing 1 tablet of complete protease inhibitor cocktail (Roche) and 0.1% b-mercapto-ethanol without detergents. Samples were than centrifugated at 3000 g for 10 minutes at 4°C. The supernatant was then centrifugated at 100,000 g for 1 hour at 4°C in a Sorvall™ WX Floor Ultra Centrifuge (Thermo Scientific, Asheville, NC, USA) with a 70.1Ti rotor. The supernatant was taken and proteins in the cytosolic fraction were solubilized by adding 1% NP-40. The remaining pellet was resuspended in 1 ml lysis buffer without b-mercapto-ethanol and centrifuged at 100,000 g for 15 minutes at 4°C to get rid of all cytosolic parts. The remaining pellet consists of the membrane fraction and is subsequently resuspended in 100 μl lysis buffer containing 1% NP-40 and b-mercapto-ethanol. To remove any debris, membrane fractions were spun a final time at 12,000 g for 10 minutes at 4°C. The remaining supernatant was used for further experiments.