Millipore polyvinylidene difluoride membranes

The protein levels of the Exos were measured using the BCA protein assay kit (Thermo Fisher Scientific, MA, USA). The ultrastructure of the Exos was inspected using a transmission electron microscope (JEM 1200-EX, Japan). In brief, Exos suspensions were loaded on 200-mesh formvar-coated grids and then negatively stained with phosphotungstic acid. The samples were observed under a transmission electron microscope at a voltage of 100 kV. The concentration and size distribution of the Exos were detected by nanoparticle tracking analysis (Nanosight NS300, Malvern, UK). Exosomal markers of CD9 and TSG101 and the negative marker of calnexin were detected by western blotting. In brief, the samples were separated by SDS-PAGE and transferred onto Millipore polyvinylidene difluoride membranes. The membranes were incubated overnight at 4°C with the primary antibodies of CD9 (Zenbio, Chengdu, China), TSG101 (Zenbio, Chengdu, China), and calnexin (Affinity Biosciences, Jiangsu, China) and visualized with enhanced chemiluminescence reagent (Millipore, MA, USA). Exos were labelled with DiI (Beyotime Biotechnology, Shanghai, China) for in vivo tracer experiments.

The total protein extracts from cells were prepared in ice-cold cell lysis buffer (50 mM Tris-HCl, pH 8.0, with 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor (500 mM phenylmethylsulfonyl fluoride). The cell homogenate was spun at 12,000 rpm for 15 min at 4°C, and the protein concentration in the supernatant was determined by the Bradford assay. Approximately 30 μg of the supernatant was resolved by SDS-PAGE and transferred onto Millipore polyvinylidene difluoride membranes. The membranes were blocked for 3 h with 10% (w/v) nonfat dry milk in 1×PBS and incubated with primary antibodies diluted in 3% milk 1×PBS buffer at 4°C overnight. After washing with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T), the blots were incubated for 1 h at RT with goat anti-rabbit/mouse IgG horseradish peroxidase-conjugated secondary antibody (1:3,000 dilution) in 3% milk 1×PBS buffer. The blots were again washed thrice with TBS-T for 15 min and then developed with ECL Western blotting detection reagent (GE Healthcare). All blots were reprobed for β-actin (1:3,000) or GAPDH (1:3,000) as an internal reference for protein loading. The band intensities of scanned blots were quantified using Image J. The integrated intensity of a fixed area was measured, and background levels were subtracted.