Carboxylated polystyrene beads

The concentration and size distribution of particles was measured with RPS (qNano; Izon Science Ltd, Christchurch, New Zealand) using an NP200A nanopore. This nanopore was suitable for the detection of 100–400 nm particles. Samples were measured with 7 mbar pressure, 45 mm stretch and 0.34 V. Samples were analysed for 5 minutes or until 1,000 vesicles were counted, whichever came first. To calibrate size and concentration, carboxylated polystyrene beads (Izon Science Ltd) were sonicated for 10 seconds, diluted in PBS with 0.3 mM sodium dodecyl sulphate and analysed immediately after dilution.

Size and concentration of isolated EVs were quantified by TRPS using the qNano (Izon Science, Oxford, UK). All samples were diluted at least two-fold with 0.03% Tween® 20 in PBS (v/v) prior to the measurement. NP200 (Izon Science, Oxford, UK) pores and a pressure of 7 or 8 mbar were used. Calibration runs were performed with 210 nm diameter carboxylated polystyrene beads (Izon Science, Oxford, UK) before and after each sample. All samples were measured two times. The system was cleaned after each measurement with PBS for 3 min at 20 mbar. Concentration and size were determined using the provided software (Izon Control Suite, version 3.2, Izon Science, Oxford, UK).

RPS (qNano, Izon Science, Christchurch, New Zealand) is a technique based on the Coulter principle allowing single-particle measurements in suspension (13 (link)). An RPS instrument equipped with NP200 nanopore (approximate detection range: 100–400 nm) was used to measure the concentration and size distribution of particles in suspension. RPS was operated at a pressure of 7 or 15 mBar depending on the particle rate (ideally >100/minute). The voltage was operated at 0.34–0.5 V and the stretch was adjusted until the baseline current of the signal trace was approximately 100 nA (14 (link)). Carboxylated polystyrene beads with concentration 2.4×10⁹/mL (203 nm, Izon Science) were used to calibrate the size and concentration following manufacturer's instructions. EV-containing samples were diluted 2- to 20-fold in PBS and measured using identical settings. The measurement time was 10 minutes or until 500 counts was reached, whichever came first. Measurement and analysis were performed using Izon Control Suite v2.2.2.44 software.