0.22 μm pore filter

Five micrometers GW4869 (Sigma-Aldrich, MO, USA) was added to the cells for culture if necessary, and exosomes were isolated from the cell culture supernatant by differential centrifugation. In brief, 2.5 × 10⁶ HCC cells were seeded in 150 mm dishes and allowed to recover for 24 h. Then, the cells were washed twice with prewarmed phosphate-buffered saline (PBS), and the culture medium was replaced with exosome-free medium supplemented with 10% exosome-depleted FBS. After 2 days of culture, the conditioned medium from cells reaching ~ 90% confluence was harvested and subjected to serial centrifugations for 10 min at 500 g and 30 min at 16500 g, followed by filtration through a 0.22 μm pore filter (Millipore). The filtrated medium was ultracentrifuged at 110000 g for 70 min to harvest exosomes. The exosome pellet was washed with PBS and then collected by ultracentrifugation at 110000 g for 70 min on a 40% w/v sucrose cushion. The floating exosomes were collected and pelleted again by ultracentrifugation at 110000 g for 70 min. High-resolution transmission electron microscopy (HR-TEM) of the exosomes was carried out with a JEOL 2010 microscope (Akishima, Japan) at 200 kV.

PEDVs were labeled with a lipophilic cationic dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD; Thermo Fisher, USA). A final concentration of 250 μM DiD dye was used to label the purified viruses, and it was first incubated at room temperature for 90 min with a soft vortex to ensure sufficient partition of DiD dye into the viral envelope (23 (link), 24 (link), 42 (link)). Next, excess dyes were removed using a NAP-10 filtration column (GE Healthcare). Finally, DiD-labeled PEDVs were filtered through a 0.22-μm pore filter (Millipore), aliquoted, and stored at −80°C.

CSE was prepared as previously described (Xu et al., 2018 (link)). Briefly, a vacuum suction device was used to draw smoke from Marlborough cigarettes (Philip Morris, United States) into glass tubes containing 10 mL of room-temperature Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, United States) at a constant speed. Each cigarette was continuously aspirated for 5 min. The resulting CSE solution was added to a 96-well Costar plate (200 mL of solution per well) and the absorbance was measured at 320 nm with a microplate reader (Spark^®, Tecan, Mannedorf, Switzerland). The optical density of the CSE was adjusted to 1.0 in DMEM, and the resultant CSE solution was considered as 100% CSE. The diluted solution was then sterilized with a 0.22 μm pore filter (Millipore, United States). The sterilized solution was further diluted in serum-free medium to the concentrations required for the experiments described below. Dilutions occurred within 1 h of sterilization.

Small extracellular vesicles derived from hucMSCs were isolated and purified as described previously (Wu et al., 2021 (link)). Cell supernatants were collected and centrifuged at 1,500 × g for 20 mins to remove cell debris and then centrifuged at 10,000 × g for 30 min. Next, the sEVs were concentrated with a 100 kDa molecular weight cutoff ultrafiltration membrane (MWCO) (Millipore, Billerica, MA, United States). Subsequently, the concentrated solution from the upper tube was collected for ultracentrifugation at 100,000 g for 70 min at 4°C to pellet the hucMSC-sEVs. Then pellet containing sEVs was resuspended in phosphate-buffered saline (PBS) and centrifuged again at 100,000 g for 70 min. Finally, the pelleted sEVs were washed and gathered from the bottom of the tube with PBS. sEVs were finally filtered with a 0.22 μm pore filter (Millipore, Billerica, Massachusetts, United States) and stored at −80°C. The diameter of the concentrated sEVs was determined by nanoparticle tracking analysis (NTA) (NanoSight, Amesbury, United Kingdom). sEVs were also identified morphologically by transmission electron microscopy (FEI Tecnai 12, Philips, Netherlands). Western blotting was also used to confirm the presence of characteristic markers of sEVs (CD81, HSP70, and Alix).

Exosomes were isolated from the conditioned medium of gastric cancer cells as previously described [29 (link)]. In brief, cells were cultured in exosome-depleted medium and the supernatants were collected after 48 h. The conditioned medium was centrifuged at 1,000 g for 10 min to remove cell debris followed by 30 min at 10,000 g using 100 KDa MWCO before the concentrated solutions were filtrated through a 0.22-μm pore filter (Millipore, Shanghai, China). Exosomes were precipitated by adding the exosome quick extraction solution (System Biosciences, Palo Alto, CA, USA) at a ratio of 1:5 at 4 ^oC for 12 h. Exosomes were dissolved with PBS and stored at -80 ^oC. Protein concentration was determined by BCA protein assay kit (ThermoFisher Scientific, Shanghai, China). The size and concentration of exosomes were measured by Nanoparticle Tracking Analysis (NTA). The morphology of purified exosomes was identified by transmission electron microscopy (Tecnai 12; Philips) and the expression of exosomal markers CD9 and CD81 by western blot.

Exosomes were isolated and purified as described previously [25 (link)]. Cell-conditioned medium with 10% FBS in which bovine exosomes and protein aggregates were removed by ultracentrifugation at 10000 ×g for 16 h at 4°C. Following 48 h culture, cell supernatants were collected and centrifuged at 2000 ×g for 20 min to remove cell debris and then centrifuged at 1000 ×g for 30 min using a 100 kDa molecular weight cutoff ultrafiltration membrane (MWCO) (Millipore, Billerica, Massachusetts, USA) to concentrate. After that, the concentrated supernatants were loaded upon a 30% sucrose/D₂O cushion and ultracentrifuged at 100000 ×g for 2 h at 4°C. Exosomes were gathered from the bottom of the tube and washed with PBS for three times by centrifugation at 1000 ×g for 30 min using a 100 kDa MWCO (Millipore, Billerica, Massachusetts, USA). Exosomes were finally filtrated on a 0.22 μm pore filter (Millipore, Billerica, Massachusetts, USA) and stored at −80°C. Concentration of concentrated exosomes was determined by nanoparticle tracking analysis (NTA) (NanoSight, Amesbury, U.K.). Exosomes were also identified by transmission electron microscopy (FEI Tecnai 12, Philips, Netherlands) for the morphology and the size and ImageStreamX Imaging Flow Cytometer (Amnis, WA, USA) for exosomal markers, CD9 and CD63.