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).