2.4. Plasma exosomes isolation, characterization and labeling
Blood was centrifuged at 2,000×g for 10 min to obtain plasma. The plasma was collected and centrifuged at 3,000×g for 15 min to remove cells and cellular debris. The supernatant was diluted in sterile phosphate-buffered saline (PBS) and centrifuged again at 12,000×g for 45 minutes followed by 2 h ultracentrifugation at 120,000×g. The pellet was diluted with PBS and filtered through a 0.22 μm filter (Millipore). The sample was then transferred to a clean tube and centrifuged again at 120,000×g for 70 min. The pellet was then collected and re-suspended in 100 μl PBS for later functional or biochemical assay. All centrifugations were performed at 4 ℃. The PBS was also filtered through a 0.22 μm filter to avoid impurity.
After isolation, exosomes were assessed by transmission electron microscopy (TEM) and NanoSight analysis (NTA). The size and morphological characteristics of the exosomes preparations were analyzed by TEM. Briefly, the exosomes pellets were fixed with 2% PFA and deposited onto formvar carbon-coated electron-microscopy grids. The grids were separately transferred into 1% glutaraldehyde and stained by adding 2% uranyl acetate. After air dry, the grids were viewed under TEM. The size and concentration of exosomes were determined by NTA. Exosomes were resuspended in PBS, and the measurement was completed by ZetaView PMX 110 (Meerbusch, Germany).
Purified exosomes were labeled with green fluorescent lipophilic linker PKH67 (Sigma, USA) by following the manufacturer’s instructions. Briefly, exosomes were resuspended in 0.5 ml Dilution C, and 4 μl PKH67 was added to the suspended exosomes, followed by 4 min incubation at room temperature. The stain reaction was stopped with 1% bovine serum albumin (BSA). After dilution with PBS, the sample was centrifuged at 120,000g for 70 min at 4˚C. The pellet was washed in PBS and again centrifuged at 120,000g for 70 min at 4˚C. Finally, the pellet was re-suspended in culture medium, and incubated with PASMCs for 12 h. After incubation, PASMCs were fixed with 4% PFA and stained with DAPI (Beyotime, China). Confocal microscopic images were captured with confocal laser microscope (LSM900, ZEISS, Germany).