br Exosomes Extraction br The exosomes in serum were
The exosomes in serum were removed by means of ultracentrifuga-tion of FBS at 100,000 g for 8 h. When BMSCs’ confluence reached around 80%, the supernatant was removed. BMSCS were cultured in 10% exosome-free FBS at 37 C in a CO2 incubator for 48 h. The collected supernatant was centrifuged in a gradual manner at varying speeds according to the following steps: 300 g for 10 min at 4 C with the removal of the precipitation, at 2,000 g for 15 min at 4 C with the Luteolin removed, at 5,000 g for 15 min at 4 C with the precipitation removed, and at 12,000 g for 30 min at 4 C following the collection of the precipitation. The supernatant was subsequently centrifuged at 12,000 g for 70 min at 4 C with the precipitation collected. The supernatant following centrifugation was centrifuged at overspeed for 70 min at 100,000 g at 4 C, after which the precipitation was collected, followed by centrifugation for 70 min at 100,000 g at 4 C with the precipitation collected.
Nanoparticles Tracking Analysis
20 mg of exosomes was dissolved in 1 mL PBS and vortexed for 1 min in order to ensure a uniform distribution. NanoSight nanoparticle tracking analyzer (Malvern Panalytical, Worcestershire, UK) was em-ployed in order to directly determine the size distribution.
Transmission Electron Microscopy Observation
The prepared exosomes were promptly fixed in 4% glutaraldehyde for fixation purposes for 2 h under 4 C conditions, fixed with 1% osmium tetroxide for 2 h, and dehydrated using conventional gradient ethanol and acetone. The exosomes were immersed, embedded, and polymer-ized with ethoxyline resin to prepare slices at a thickness of 0.5 mm. After positioning under a light microscope, ultrathin slices with a
Table 2. Primer Sequence for qRT-PCR
F 50 -GCTGTCTTGCCACAGACCC
R 50 -TGGAATATTAAGAAGGCAGTT
R 50 -ATGAGGTCCACCACCCTGTT-30
ADAM9, a disintegrin and a metalloproteinase-9; F, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR-126-3p, microRNA-126-3p; R, reverse.
thickness of 60 mm were prepared, stained with uranium acetate and lead citron citrate, and observed under an electron microscope.
Acetylcholinesterase Activity Assay
Exosomes extracted using multistep ultracentrifugation were diluted with PBS to 110 mL. Cells in each well in the 96-well plate were added with a solution containing PBS and 5,50-dithiobis-2-nitrobenzoic acid (DTNB) solution (0.1 mmol/L) with equal volume (37.5 mL); then the acetylcholine solution of sulfur iodide (1.25 mmol/L) was added to a final volume of 300 mL. After 30 min, the optical density (OD) values of each well were detected using a microplate reader at a wavelength of 412 nm.
Co-culture of Pancreatic Cancer Cells and BMSCs
The pancreatic cancer cell lines PANC-1 and BMSCs were attached with trypsin, centrifuged at 1,000 g for 5 min, and resuspended with 3 mL DMEM culture medium. Next, 1 mL suspension was diluted 20 times more. The diluted cell suspension was fully mixed, and 10 mL resuspension was counted under the cell count plate. Finally, PANC-1 and BMSCs, at the ratio of 3:1, were spread to a diameter of 0.4 mm co-culture chamber. In the co-culture chamber, BMSCs (about 0.4 105) were placed into the basolateral chamber, whereas PANA-1 cells (about 1.2 105) were placed into the apical chamber, with the co-culture chamber placed in the six-well plate. The PANC-1 cells in the apical chamber were cultured with DMEM containing 10% serum, and BMSCs cells in the basolateral chamber were cultured in DMEM culture medium containing 15% serum for 4–5 days with the liquid and medium in both apical and basolateral chambers changed every 1–2 days. After co-culturing, the apical chamber was withdrawn and the cells were used for subse-quent experiments.
Confocal Fluorescence Microscopy
BMSCs transfected with miR-126-3p-Cy3 (GenePharma, Shanghai, China) carrying red fluorescent markers were co-cultured with pancreatic cancer cells transfected with pCDNA3.1-GFP. The uptake of the exosomes by pancreatic cancer cells was observed under a
confocal fluorescence microscope after 6, 24, and 48 h of co-culture, respectively. After 2 days of co-culture, two cell lines were separated by flow cytometry.
RNA Isolation and Quantitation