Extracellular vesicle measurements with nanoparticle tracking analysis – An accuracy and repeatability comparison between NanoSight NS300 and ZetaView

Abstract

The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% D TEM range: 79.5–134.3) compared to ZetaView (% D TEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution.

Extracellular Vesicles from the Apoplastic Fungal Wheat Pathogen Zymoseptoria Tritici

“…1c). The apparent lack of smaller particles represented in the NTA size distribution when compared to the TEM measurements, may be artefactual of NTA systems' known underreporting of particles < 50 nm and/or the dehydration and shrinkage caused by negative staining of EVs for TEM [21,33]. Z. tritici EVs samples typically had a concentration of 0.12-0.15 µg of protein and 1.45-2.43 ×108 particles per mL of culture filtrate.…”

Nanoparticles in 472 Human Cerebrospinal Fluid: Changes in Extracellular Vesicle Concentration and miR-21 Expression as a Biomarker for Leptomeningeal metastasis

“…Quantitative measurement of EVs were tested with ExoView Tetraspanin kits (NanoView Bioscience, Boston, MA) as previously decribed [22]. CSF was diluted in PBS with 0.05% Tween-20 (PBST) and incubated on the ExoView Tetraspanin Chip (EV-TC-TTS-01) placed in a sealed 24-well plate for overnight at room temperature.…”

Exosomal Thrombospondin-1 Disrupts the Integrity of Endothelial Intercellular Junctions to Facilitate Breast Cancer Cell Metastasis

“…After being treated with 10 µL of 4% glutaraldehyde and then stained with 10 µL of 2% sodium phosphotungstate, the grids with fixed exosomes were air-dried and visualized at 120 kV under a Tecnai G2 F30 transmission electron microscope (FEI, Hillsboro, OR, USA). The size distribution and concentration of exosomes were determined using a ZetaView PMX-120 (Particle Metrix, Meerbusch, Germany), which was equipped with a fast video capture system and a ZetaView software for nanoparticle tracking analysis [35].…”