Malvern ns300

According to previous methods [24 (link)], exosomes were separated using differential centrifugation. BMMSCs or HO-1/BMMSCs were cultured in exosome-free FBS media (Sesh-biotech, Shanghai, China) for 72 h, and the supernatant was collected and centrifuged at 250×g for 15 min and 3000×g for 30 min, consecutively, to remove cell fragments. After 0.22 μm filtration and centrifugation at 110,000×g for 140 min at 4 ℃, the exosomes were deposited at the bottom of the ultracentrifuge tube. The exosomes were re-suspended in sterile phosphate-buffered saline (PBS), and the supernatant was retained after centrifugation at 10,000×g for 5 min. The obtained exosomes were temporarily stored at − 80 ℃. Western blotting was used to detect exosomes markers, and antibodies against CD9, tumor susceptibility 101 (TSG101), CD63, Calnexin (all ProteinTech, Wuhan, China), and CD81 (Abcam, Cambridge, UK) were used. The morphology of the exosomes was observed using transmission electron microscopy (TEM, Hitachi-HT7700, Tokyo, Japan). The particle concentration, size, and distribution were detected using nanoparticle tracking analysis (NTA, Malvern-NS300, Malvern, UK).

Particle concentration and mode particle size was measured by Malvern NS300 (Malvern Instruments Ltd, Malvern, UK) with a 532 nm green laser and Scientific CMOS trigger camera. Nanosight NTA 2.3.5 was used for data processing.
Based on expected polydispersity, video length was set to 60 seconds for 10-40 particles per frame. The camera was set to 12, shutter set to 600, gain to 350, and detection threshold to 3. Sample viscosity was set to water and temperature to 23.3°C. The default settings were used for minimum expected particle size, minimum track length, and blur. Samples for comparison were run within the same session to minimize variation.
Samples were diluted in cell culture grade 1× PBS to obtain approximately 30 particles per frame. Samples were loaded into the sample chamber using a 1 mL Monoject tuberculin syringe (Covidien, Mansfield MA) attached to a syringe pump (Harvard Apparatus, Holliston MA). Flow rate for advancing the sample and agitating between replicates was 255 µL per minute and readings were taken at a flow rate of 25 µL per minute. Four replicate 60 second sessions were taken per syringe for each patient. Data are expressed as mean ± standard error of the mean.

After 72h of HO-1/BMMSC culture in exosome-free serum medium, debris and dead cells in the medium were removed by centrifugation at 250×g for 15min and 3000×g for 30min and then filtered through a 0.22μm filter. The medium was then subjected to ultracentrifugation at 110,000×g for 140min at 4°C. Exosomes were resuspended in sterile phosphate-buffered saline (PBS).
Exosome morphology was observed using a transmission electron microscope (Hitachi-HT7700, Tokyo, Japan). The exosomal particle size was determined using nanoparticle tracking analysis (Malvern-NS300, Malvern, UK). The levels of exosome-specific proteins CD9, CD63, tumor susceptibility 101 (TSG101), and calnexin (negative control; ProteinTech, Wuhan, China) were detected using western blotting.
To monitor exosome trafficking, exosomes were labeled with chloromethyl-1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (CM-DiI fluorescent dye, Invitrogen, Waltham, MA, USA) for 15 min in the dark at 4°C. After CM-DiI staining, the exosomes were washed in PBS and collected by ultracentrifugation (110,000×g for 70min) at 4°C. Finally, CM-DiI-labeled exosomes were resuspended in PBS. The rat liver cell line IAR20 was cocultured with the labeled exosomes for 6 h, and the uptake of exosomes was observed using a confocal microscope (FluoView™-FV1000, Olympus, Tokyo, Japan).