Nanoparticle free water

Extracellular vesicles present in urinary samples were analyzed by nanoparticle tracking using the NanoSight LM10 system (NanoSight Ltd, Amesbury, Wiltshire, UK) configured with a 405 nm laser and a high sensitivity digital camera system (OrcaFlash 2.8; Hamamatsu C11440, NanoSight Ltd). Videos of 60 s were collected and analyzed using NTA-software (version 2.2) with the minimal expected particle size set to 30 nm, and minimum track length and blur set to automatic. Each sample was diluted in nanoparticle-free water (Fresenius Kabi, Runcorn, Cheshire, UK) to a concentration of between 2 × 10⁸ and 9 × 10⁸ particles/mL. This was carried out in triplicate for pre-ultracentrifuged urine, and for 4 replicates following 200,000 g ultracentrifugation.

Liposomes were characterized for diameter, polydispersity index, conductivity, electrophoretic mobility, and charge (ζ potential) using a Zetasizer Nano ZS series ZEN3600 fitted with a 633 nm laser (Malvern Instruments Ltd.). Liposomes (50 μl) containing 15-HETE-PE, generated as above, were added to 950 μl deionized water and placed in a clear plastic ζ cell (DTS 1070, Malvern) and equilibrated for 2 minutes at 25°C prior to measurement. Each was measured at least 4 times. Liposome diameter was also measured using by nanoparticle tracking analysis using a NanoSight LM10 system (Malvern Instruments Ltd.) running NTA-software v2.3 and configured with a temperature controlled, 488nm LM14 laser module and a high-sensitivity camera system (OrcaFlash2.8, Hamamatsu C11440). In brief, 4-mM liposomes were diluted 1,000 times in nano-particle free water (Fresenius Kabi) and analyzed under controlled fluid flow at 37°C, as previously described (49 (link)). Data were combined from 4 preparations done on different days, with each analyzed 5 times.

NTA was carried out using a NanoSight LM10 equipped with a blue laser, a CCD camera, and a syringe pump system (Malvern Instruments, UK). Prior to sEV analysis, instrument performance was assessed using 100 nm latex beads (Malvern Instruments). Samples were diluted using nanoparticle free water (Fresenius Kabi, Runcorn, UK) so that the particle concentration was within the linear range of the instrument. Samples were administered and recorded under controlled flow from a syringe pump (set to a speed of 50 arbitrary units). Six replicate videos of 30 s were recorded along with sample temperature for each specimen. Videos were batch analyzed using the integrated NTA 3.1 software, with the cameras sensitivity and detection threshold set to between 14-16 and 1-3, respectively. Particle number was calculated based upon the area under the histogram and modal and mean particle sizes were determined. Background measurements of culture media that had not been exposed to cells and PBS used to dilute sEVs contained negligible particles.