Cpc200

Tunable resistive pulse sensing (TRPS) analysis of the size distribution and concentration of nanoparticles in the CD81+ pool of SEC fractions was conducted on a qNano Gold platform (Izon Science, Christchurch, New Zealand). The nanopore was wetted, coated and equilibrated with an Izon reagent kit as per the manufacturer’s instructions. The calibration was performed with 200nm carboxylated polystyrene beads (CPC200, Izon Science, Christchurch, New Zealand), diluted 500-fold in filtered electrolyte solution, and stretch was adjusted until the blockade magnitude for calibration particles averaged between 0.25 and 0.3 nA. Measurements were conducted at a constant 46.00 mm stretch and a baseline current at approximately 125 nA. Samples were diluted twofold in filtered electrolyte solution and vortexed for 30 s prior to TRPS measurements. Each sample was measured at two pressure steps of 5 and 2.5 mbar, in triplicate. At least 500 particles were recorded for each measurement. Before each measurement, the nanopore was washed with the electrolyte solution at a pressure of 20 mbar to avoid cross-contamination between samples. Data processing and analysis were performed in Izon Control Suite v3.4 software (Izon Science, Christchurch, New Zealand). A concentration fraction from 110 to 420 nm was applied for all measurements prior to any data analysis.

Following isolation, ADNVs in PBS were quantified through tunable resistive pulse sensing (qNANO Gold, Izon Science Ltd., Cambridge, MA, USA). The analysis also provided data on size distribution of particles in the ADNV fraction. The nanopore (NP150, Izon Science Ltd., Cambridge, MA, USA) was stretched 49 mm wide using a digital caliber. The preparation of the instrument was performed with reagents provided by the manufacturer following the manufacturer’s instructions. Each sample was analyzed at two pressure points, 10 atm and 20 atm. During each measurement, the particle rate was maintained above 200 particles/min, and the total particle count surpassed 500 particles. Calibration particle (CPC200, Izon Science Ltd., Cambridge, MA, USA) measurements were taken at both 10 atm and 20 atm and were used for calibrating each sample measurement during data analysis. The protein content was also established through Bradford assay with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. Absorbance was measured at 570 nm using multilabel plate reader Victor 3 (Perkin Elmer, Milano, Italy).

The size and concentration of EVs were measured using a tunable resistive pulse sensing (TRPS) method (qNano, Izon Science). Samples were filtered before processed in the qNano instrument using a 1-μm syringe filter (Tisch Scientific, North Bend, OH). 45 μL of biofluid was pipetted into different nanopore stretchable membranes (NP150, NP300, NP600, NP800, and NP1000 from Izon Science) to cover the wide size range of EVs^{36 (link)}. A pressure of 1 kPa and different voltages (0.38 V, 0.32 V, 0.26 V, 0.18 V and 0.12 V) were applied. Polystyrene nanoparticles of different known sizes and concentrations were used for calibration (CPC70, 70 nm; CPC100, 100 nm; CPC200, 200 nm; CPC400, 400 nm; CPC800, 800 nm; CPC1000, 1000 nm from Izon Science, Medford, MA). EVs with a size range of 70–1000 nm were characterized.

Quantification and size characterization of EVs were measured using a tunable resistive pulse sensing (TRPS) instrument (Exoid; IZON Science Ltd, Christchurch, New Zealand). Two different nanopores (NP250, NP400, IZON Science Ltd.) were used to assessment in the size range. Carboxylate polystyrene calibration particles (CPC200 and CPC 500, IZON Science Ltd.) were used with NP200 and NP400 nanopores to ensure optimization conditions (e.g., size, concentration). All calibrations and sample measurements were run under the same conditions recommended by the manufacturer and a minimum of 500 particles was recorded at three different pressures. The acquired data was analyzed using Izon Control Suite software (Izon Control Suite version 3.2.2.268, Izon Science Ltd.).

VLP size distribution, particle concentration, and charge (zeta potential) were evaluated using TRPS. These measurements were performed with the qNano (Izon Sciences) equipment and nanopore membranes NP200 (Izon Sciences), rated for particles between 80 and 500 nm. The instrument was set up and calibrated according to the manufacturer’s instructions. To calibrate size and particle concentration, carboxylated polystyrene particles with a mode diameter of 340 nm (SKP400E, Izon Science) with a concentration of 6 × 10¹⁰ particles/ml were used. All measurements were done using a membrane stretch of 45 mm, a voltage of 0.20 V, and a pressure of 5 mbar. The measurement duration was dependent on the number of particles detected with a minimum of 500 events. Zeta potential measurements were performed using PBS buffer using calibration particles with a diameter of 220 nm (CPC 200, Izon Science) and a nanopore membrane NP200 (Izon Science). The selected membrane stretch was 45.30 mm. The pressure (P) and voltage (V) calibration points were selected as V1P1, V1P2, V2P1, and V3P1 with V1, V2, and V3 as 0.32, 0.26, and 0.29 V, respectively, and P1 and P2 as 1 and 2 mbar. The zeta potential of VLP samples was measured using V1P1 voltage and pressure setting.

Quantification and size characterization of EVs in RBCs were measured using a tunable resistive pulse sensing instrument (TRPS/qNano system; IZON Science Ltd, Medford, MA, USA) as previously described in detail [13, 14]. Two different nanopores (NP200 and NP400) were used to target EVs <1.0 µm in size using a standard stretch range (43–47 mm). Carboxylate polystyrene calibration particles (CPC200; IZON Science Ltd) were used with the NP200 to characterize EVs less than 200 nm in diameter, while CPC500 (IZON Science Ltd.) were used with the NP400 to calibrate for EVs ≥200 nm. Supernatant samples were diluted with electrolyte solution (Measurement Electrolyte, IZON Reagent kit, RK1) and the sample dilution was adjusted to target a particle rate of 1000–2000/min. Samples were filtered using a 0.80 μm Millex syringe filter (Merck Millipore Ltd) before being analysed with NP400 or NP200 as recommended by the manufacturer. Samples and calibration particles measurements were run under the same conditions and at least 1000 particles were recorded with two different standard pressure ranges (1 unit = 1 mbar). Data obtained were analysed using IZON Control Suite software Version 3.3.