Flow nanoanalyzer

MVECs were grown in exosome-free medium. After clinorotation for 48 h, exosomes were extracted via ultracentrifugation from cell supernatants. Briefly, the harvested culture supernatant was centrifuged at 300× g for 10 min at 4 °C, then again at 2000× g for 10 min, and finally at 10,000× g for 30 min to eliminate cellular debris and dead cells from the medium. Then, the collected supernatant was ultracentrifuged (Beckman Coulter, Brea, CA, USA) at 100,000× g for 70 min. To remove contaminating proteins, the exosomes were washed with PBS and subsequently ultracentrifuged at 100,000× g for another 70 min. After carefully removing the supernatant, the pellets were resuspended in PBS and kept at −80 °C until use. The protein concentration in the exosome suspension was detected by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Flow NanoAnalyzer (NanoFCM, Xiamen, China) was implemented to evaluate the size distribution of isolated exosomes. TEM (Hitachi, Tokyo, Japan) was applied to examine the morphology of the extracted exosomes.

To determine size distribution, exosomes were resuspended with PBS after ultracentrifugation and detected with a Flow NanoAnalyzer (NanoFCM, China).

Nanoflow cytometry was used to determine the molecular marker expression and the concentration and size distribution of the EVs on a Flow NanoAnalyzer (N30E, NanoFCM INC., Fujian, China). For concentration and size distribution analysis, EV samples were diluted 1 : 100 in PBS and directly analyzed according to the Flow NanoAnalyzer's instructions. For molecular markers, EV samples were diluted 1 : 100 in PBS and labeled by fluorescent antibodies, including FITC Mouse Anti-Human CD9 (Cat# 555371, BD Biosciences, NJ, USA), FITC Mouse Anti-Human CD63 (Cat# 556019, BD Biosciences), FITC Mouse Anti-Human CD81 (Cat# 551108, BD Biosciences), and FITC Mouse IgG (Cat# 400108, BioLegend, CA, USA).

The size of the exosomes was analyzed using a Flow NanoAnalyzer (NanoFCM) equipped with nanoparticle particle tracking software. According to the manufacturer’s recommendation, the samples were illuminated with the laser, and the movement of nanoparticles due to Brownian motion was recorded for 60 s at a mean frame rate of 20 frames/s.

High-resolution flowcytometry for nanoparticle analyses were performed using the Flow Nanoanalyzer (N30, NanoFCM, Xiamen, China). In details, 100 μL of isolated EV sample was mixed with 4 μL of Alexa488 anti-human TNFRSF10B antibody (FAB6311G, R&D Systems, Minneapolis, MN) and 2 μL of PE anti-human CD63 antibody (561925, BD Biosciences, Franklin Lakes, NJ) and incubated at 37 °C for 30 minutes. Unlabeled antibodies were removed using second SEC with the qEVsingle column (70 nm Gen 2, IZON, Medford, MA). Again, labeled EVs were recovered in the first elution peak and then subsequently subjected to nanoparticle data collections. All data were converted to FCS 3.0 using NF Profession (Version 1.0, NanoFCM) and analyzed with FlowJo (Version 10.8.1, BD, Franklin Lakes, NJ).