Zetaview nanoparticle tracking analyzer

The hydrodynamic size distribution of the EV, this is, the particle concentration as a function of the diameter was obtained by Nanoparticle Tracking Analysis (NTA). A NanoSight LM10-HS(GB) FT14 (NanoSight, Amesbury, United Kingdom) equipped with a sample chamber with a 405 nm laser and a high-sensitivity EMCCD camera was used. Video images of the Brownian motion of the particles were captured and analyzed by the NTA 2.3 image analysis software. All samples were measured at least in triplicate at 25°C, with manual shutter, gain, brightness, and threshold adjustments. In selected samples NTA was performed using the ZetaView Nanoparticle Tracking Analyzer (Particle Metrix, RRID : SCR_016647). Samples were measured for size and concentration in scatter mode (488 nm laser) and standard instrument settings following the manufacturer’s instructions.
In addition, the number of particles was also assessed in selected samples using the Bio-Rad ZE5 Cell Analyzer Flow Cytometer (RRID : SCR_019713) that has small particle detection capability (forward scatter (FSC) from the 405 nm laser).

Samples were processed in duplicate and diluted with PBS over a range of concentrations to obtain between 10 and 100 particles per image before analysis. Samples were added to a chamber using a ZetaView Nanoparticle Tracking Analyzer (Particle Metrix, Germany) to automatically measure the average diameter and concentration.

Nanoparticle tracking analysis (NTA) of exosome samples was conducted by the ZetaView Nanoparticle Tracking Analyzer (Particle Metrix, Meerbusch, Germany) following the standard protocols [32 (link)]. Exosomes isolated through ultracentrifugation were diluted in PBS (1:100) and loaded into the flushed chamber. Analyses were automatically carried out, and the concentration and diameter of exosomes were then measured and analyzed through the corresponding software ZetaView 8.03.04.01.
Exosomes bonded on the surface of microbeads were characterized by scanning electron microscope (SEM) [33 (link)]. Approximately 1000 beads were mixed with 1 mL of cell culture supernatant. After incubation at 37 °C for 20 min, the mixture was centrifuged at 1000×g for 10 min and resuspended in 100 µL of PBS. The samples were fixed in 4% paraformaldehyde for 1 h, dehydrated in a series of increasing ethanol concentrations, uniformly spread onto a silica glass, and then lyophilized overnight. After that, the silica glass was vacuumized and sputter-coated with gold at room temperature for 60 s. Finally, the morphology of exosomes on microbeads was examined under a field emission scanning electron microscope (JSM-7800F; JEOL, Tokyo, Japan).

Particle size and number of EV solutions were determined in a ZetaView Nanoparticle Tracking Analyzer (Particle Metrix, software ZetaView 8-2-31) as described [21 (link)].

The size and Zeta-potential of the exosomes were measured using ZetaView® nanoparticle tracking analyzer (Particle Metrix GmbH, Meerbusch, Germany).

The size distribution and concentration of RBCEVs were quantified using a ZetaView^® nanoparticle tracking analyzer (Particle Metrix, Germany). Because hemoglobin is the major constituent of RBCEVs, RBCEV quantity is indicated by hemoglobin quantity throughout this study. The hemoglobin contents of RBCEVs were quantified using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific).