Zetaview pmx120 instrument

Dynamic light scattering (DLS) measurements were performed at room temperature with an equipment consisting of a goniometer system (ALV GmbH, Langen, Germany), a diode-pumped solid-state laser light source (Melles Griot, IDEX corp., Lake Forest, IL, USA; 58-BLS-301, 457 nm, 150 mW), and a light detector (Hamamatsu, Japan; H7155 PMT module). The evaluation software yielded the autocorrelation function of scattered light intensity, which was further analyzed by the maximum entropy method, from where the different contributions of this function were determined. The radius of the particles was calculated using sphere approximation.^{36 (link)}For nanoparticle tracking analysis (NTA), samples were resuspended in 1 ml of PBS to reach appropriate particle concentration range for the measurement. Particle size distribution and concentration were analyzed on ZetaView PMX120 instrument (Particle Metrix, Inning am Ammersee, Germany). For each measurement, 11 cell positions were scanned at 25°C (in 2 cycles) with the following camera settings: shutter speed—100, sensitivity—75, frame rate—7.5, video quality—medium (30 frames). The videos were analyzed by the ZetaView Analyze software 8.05.10 with a minimum area of 5, maximum area of 1,000, and a minimum brightness of 20.

Size distribution and concentration of MC‐derived EVs in the conditioned medium or in the case of separated EVs were determined by tuneable resistive pulse sensing (TRPS) using a qNano instrument (IZON Science) as we described previously (Osteikoetxea et al., 2015). Briefly, EV samples from 10 ml supernatant of 20 × 10⁶ cells were diluted in 0.2 μm‐filtered 0.01% Triton X‐100 (Sigma) containing PBS (in 1:50 and 1:100). At least 500 particles were counted using 3–5 mbar pressure using NP200, NP400, NP800 and NP2000 nanopore membranes. Calibration was performed with known concentration of beads CPC100, CPC200, CPC300, CPC800, CPC1000 and CPC2000 (diluted as suggested by IZON user manual) in 0.2 μm‐filtered PBS. Results were evaluated using the IZON Control Suite 3.2 software.
In some experiments, nanoparticle tracking analysis (NTA) was also used. Briefly, measurements were performed on a ZetaView PMX120 instrument (Particle Metrix, Germany). Separated sEV samples were diluted in PBS to achieve the optimal concentration (50–200 particles/frame). Each sample was measured in 11 positions (2 cycles per position) and the acquired video has been used for particle size and concentration analysis. The following camera parameters were used: shutter speed‐100, sensitivity‐80, frame rate‐30. The recordings were performed at 25C.