Zetaview analyze software

EV size distribution and concentration were determined using a ZetaView PMX-120 nanoparticle analyzer (Particle Metrix, Inning am Ammersee, Germany) equipped with Zetaview Analyze Software version 8.05.12. Prior to measurement, the system was calibrated as per manufacturer’s instructions with 100nm Nanospheres 3100A (Thermo Fisher Scientific). Measurements were performed in scatter mode, and for all measurements, the cell temperature was maintained at 25°C. Each sample was diluted in PBS to a final volume of 1 ml. Capture settings were sensitivity 80, shutter 100, and frame rate 30. Post-acquisition settings were minimum trace length 10, min brightness 30, min area 5, and max area 1000.

The EV samples were diluted to 1mL in PBS, and both particle concentration and size distribution were measured using a ZetaView PMX120 NTA instrument (Particle Metrix GmbH, Inning am Ammersee, Germany) For each measurement, 11 positions (2 cycles per position) were scanned at 25 °C. The following settings were used: auto expose; shutter speed—100; gain—28.8; offset—0; sensitivity—80 for sEVs and 75 for mEVs; frame rate—30 for sEVs and 7.5 for mEVs. The recorded videos were analyzed with a minimum area of 5, a maximum area of 1000 and a minimum brightness of 20 by ZetaView Analyze software 8.05.10 (Particle Metrix GmbH, Inning am Ammersee, Germany) [32 (link)]. When comparing the EV release from the different experimental groups, the cells were cultured under identical conditions (medium volume; tissue culture flask surface area).