Size distribution and concentration of H9c2-derived mEVs and DOPC liposomes were determined by tuneable resistive pulse-sensing analysis using a qNano instrument (IZON Science) as described previously [19 ]. In case of mEVs, twofold dilution of the sample (derived from 24 mL cell supernatant) was prepared in 0.2 μm filtered PBS. For DOPC liposomes, the DOPC liposome standard (with 1 mg/mL DOPC concentration) was 10-fold diluted with 0.2 μm filtered PBS and filtered with a 0.8 μm mesh filter (Millipore). Both samples were measured by qNano. At least 700 particles were counted using 10 mbar pressure (mEV) and 3 mbar pressure (DOPC liposomes) using a NP400 nanopore membrane. Calibration was performed with known concentration of beads CPC400G (mean diameter: 340 nm, purchased from IZON), diluted 1:1000 in 0.2 μm filtered PBS. Results were evaluated using IZON Control Suite 3.2 software.
Control suite 3
Manufactured by Izon Science
Sourced in New Zealand
Control Suite 3.2 Software is a data analysis and instrument control software developed by Izon Science. It provides a user interface for configuring and controlling Izon's analytical instruments, as well as tools for processing and visualizing the data generated by those instruments.
Automatically generated - may contain errors
Lab products found in correlation
Qnano gold, by Izon Science (5 mentions)
Skp200 calibration beads, by Izon Science (3 mentions)
Qnano, by Izon Science (2 mentions)
Cpc400g, by Izon Science (1 mentions)
Solution q, by Izon Science (1 mentions)
Cpc100b, by Izon Science (1 mentions)
Cpc800d, by Izon Science (1 mentions)
Particle free pbs, by Thermo Fisher Scientific (1 mentions)
Cpc100 calibration beads, by Izon Science (1 mentions)
7 protocols using control suite 3
1
Characterization of mEVs and Liposomes
2
Extracellular Vesicle Size Analysis
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Size distribution was measured using TRPS (qNano gold, Izon) with Izon Control Suite 3.2 Software and SKP200 calibration beads (Izon, Chirstchurch, New Zealand) using NP200 nanopores. The current was adjusted to 125 nA. Where possible, at least 500 EV counts were acquired per sample. Samples used for TRPS were freshly prepared by differential ultracentrifugation followed by sucrose gradient purification. Samples were diluted with solution Q.
3
Cryo-TEM and TRPS Analysis of EVs
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The morphology and size of the isolated EVs were visualized by cryo-transmission electron microscopy (cryo-TEM) as described previously [13 ]. Additionally, the EV size distribution and concentration were determined by TRPS. TRPS was performed using a qNano Gold with Izon Control Suite 3.2 Software and SKP200 calibration beads (Izon, Chirstchurch, New Zealand). EV isolates were analysed after a single freeze-thaw cycle at −80°C. Where possible, at least 500 blockades were detected per sample. To enable comparability between the different recordings, the pore stretch was adjusted to obtain a relative particle size of 0.65 for the SKP200 calibration beads, and the voltage was adjusted to obtain a current between 125 and 130 nA. A size filter from 80 to 250 nm was applied for determining EV concentrations.
4
Optimized Nanoparticle Tracking Analysis
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TRPS was performed using a qNano Gold with Izon Control Suite 3.2 Software, an NP150 nanopore and SKP200 calibration beads (Izon, Chirstchurch, New Zealand). Cell-depleted unfiltered media and EV isolates were analysed after a single freeze-thaw cycle at −80 °C. Media were diluted 2-fold and EV isolates up to 20-fold in Solution Q (Izon). To improve comparability between measurements, the following settings were used: For UF-SEC media and isolates, the stretch of the NP150 nanopore was adjusted to obtain a relative particle size of approximately 0.65 for the SKP200 calibration beads. A concentration fraction from 80 to 250 nm was applied to these measurements. For UC and UC-wash media and isolates, the relative particle size for the SKP200 calibration beads was adjusted to 0.25, as smaller stretches resulted in pore obstruction. A concentration fraction from 110 to 300 nm was applied to these measurements. For all measurements, the voltage was adjusted to obtain a current between 120 and 130 nA. EV measurements of the dilute media were stopped after 10 minutes, while measurements of the more concentrated EV isolates were stopped after detecting 500 blockades.
5
EV size characterization by TRPS
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Size-based EV fractions released by Jurkat cells of different functional states were submitted to tunable resistive pulse sensing (TRPS) analysis using a qNano instrument (IZON Science, Cambridge, MA) as described previously59 (link), 61 (link). Briefly, serial dilutions were prepared in 0.2 µm filtered PBS from each EV fraction (derived from 30 mL cell supernatant) and measured by qNano. Particle numbers were counted for at least 3 min using 5 mbar pressure and NP100, NP800 and NP2000 nanopore membranes stretched between 45 and 47 mm. Voltage was applied between 0.1 and 0.4 V in order to achieve a stable 120 nA current. Particle size histograms were recorded when root mean square noise was below 12 pA, particle rate in time was linear, and at least 500 events were counted. Calibration was performed using known concentration of beads CPC100B (mode diameter: 110 nm), CPC800D (mode diameter: 740 nm) and CPC1000E (mode diameter: 900 nm) (all from IZON) diluted 1:1,000 in 0.2 µm filtered PBS. Results were evaluated using IZON Control Suite 3.2 software.
6
Vesicle Size Characterization by NTA and TRPS
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Vesicle size distribution was determined by nanoparticle tracking analysis (NTA) using a Malvern NanoSight NS300 (Malvern Technologies, Malvern, UK) with a high-sensitivity sCMOS camera and a 488 nm laser [28 ]. Platelet EV suspensions were diluted 1:200 in particle-free PBS (Gibco, Waltham, MA, USA) to an acceptable concentration according to manufacturer’s instructions. Samples were analysed under constant flow conditions (flow rate = 50) at 25°C. Fifteen 60 s videos were captured with a camera level of 16. Data were analysed using NTA 3.1.54 software with a detection threshold of 5. Alternatively, tunable resistive pulse sensing (TRPS) was performed using a qNano Gold with Izon Control Suite 3.2 Software and SKP400 calibration beads and an NP300 nanopore (Izon, Christchurch, New Zealand). Platelet EV suspensions were analysed after a single freeze–thaw cycle at −80°C. The NP300 nanopore was used at a stretch of approximately 45 mm and a pressure of 12 mbar, keeping the current between 120 and 130 nA for all measurements. Recordings were stopped after detection of 500 blockades. A concentration fraction from 140 to 600 nm was applied prior to determining the particle concentration.
7
Extracellular Vesicle Characterization by TRPS
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TRPS was performed using a qNano Gold with Izon Control Suite 3.2 Software and CPC100 calibration beads (Izon, Chirstchurch, New Zealand). Cell-depleted unfiltered medium was analyzed after a single freeze-thaw cycle at -80 °C. An NP150 nanopore (Izon) was coated using the Izon reagent kit for EV analysis according to the manufacturer's instructions. To obtain a stable baseline current, samples were diluted 1:2 in Solution Q (Izon) and to prevent frequent pore obstruction, 10% (v/v) Solution G (Izon) was added. The NP150 nanopore was used at a stretch of 45-47 mm and a pressure of 10-12 mbar, keeping the current between 120 and 130 nA for all measurements. Recordings were stopped after detection of 500 blockades or after 10 min.
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