Nanosight ns500 system

The size distribution of sEVs was determined by nanoparticle tracking analysis (NTA) with the NanoSight NS500 system (Malvern Instruments, Malvern, United Kingdom), and the morphology of sEVs was observed by transmission electron microscopy (TEM, Hitachi, Tokyo, Japan) as previously described (Wu et al., 2021 (link)). Western blotting was used to detect the expression of sEV-specific surface markers, including CD63, CD81, and TSG101.
The uptake of sEVs by BMSCs and HUVECs was examined by labeling sEVs with the fluorescent dye PKH26 or PKH67 (Sigma-Aldrich, Darmstadt, Germany), according to the manufacturer’s instructions, which were then incubated with BMSCs or HUVECs at 37°C for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde, stained with DAPI for 10 min, and observed by confocal microscopy (Nikon, Tokyo, Japan).

Average size distribution and particle concentration analyses of the EV-enriched isolates were performed using a NanoSight NS500 system (Malvern Instruments Ltd., Malvern, UK). Brownian motion of the particles was captured at 30 frames/s speed. For this, EV samples were diluted using filtered Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma-Aldrich, Cat. #D8537), loaded onto the NTA using a NanoSight syringe pump, and videos of the particles were recorded and analyzed using NTA version 3.3 software. Aliquots of the same filtered PBS were used as the control.

The hydrodynamic diameters of the extracted EVs were determined using nanoparticle tracking analysis (NTA) with a Nanosight NS500 system (Malvern Instruments, Malvern, UK), following the manufacturer’s guidelines. Before analysis, samples were diluted with water to achieve an approximate count of 20–50 particles per frame for particle size measurements, and 100–200 particles per frame for zeta potential assessment. For particle size measurements, the autofocus was adjusted to ensure particle clarity. Ten videos of particle motion resulting from Brownian motion were recorded for each sample, each lasting 60 s, at a temperature of 23 °C. For the zeta potential assessment, the manufacturer’s integrated automated algorithm was used. During each measurement, three cycles were executed. Within each cycle, 11 cell positions were scanned, capturing 60 frames per position. The video setting was on “high,” with the following specific settings: focus set to autofocus, camera sensitivity at 92.0, shutter speed at 70, scattering intensity at 4.0, and a cell temperature of 23 °C. The recorded video data were subsequently processed using NTA analysis software version 2.2.

Nanoparticle tracking analysis (NTA) was done using Nanosight NS 500 system (Malvern Instruments, Malvern, UK) to measure the size distribution and concentration of EVs. EV samples were gently resuspended and diluted with pre-filtered PBS according to the manufacturer’s recommended concentration range (25–100 particles/frame). To ensure consistency, identical settings were used for all measurements and the mean values were calculated after the quintuplet replicates.