The analysis was performed using a ZetaView® PMX-120 (Particle Metrix GmbH, Inning am Ammersee, Germany) nanoparticle tracking analysis (NTA) system, equipped with a light source wavelength of 488 nm. Before the measurements, the samples were diluted ten times in PBS to avoid changes in the osmolarity of the dispersion and to have an EV concentration suitable for the NTA analysis. After optimization of the instrumental parameters, the sensitivity and the shutter, set in scatter mode, were set at 70 and 100, respectively, in 3 × 33 videos of 1 s, recording a total number of particles of between 2000 and 3600 per analysis. In the fluorescent mode, the sensitivity and the shutter were set at 80 and 100, respectively. To stain the EVs, 1 uL of CellMaskTM Green (CMG) was added to the secretome and left in the dark at RT for 1 h, then the samples were diluted ten times in PBS. As a control, cell culture media with added CMG was used; no particles were detected under the same instrumental conditions.
Zetaview pmx 120
Manufactured by Particle Metrix
Sourced in Germany
The ZetaView PMX 120 is a particle characterization instrument that utilizes Nanoparticle Tracking Analysis (NTA) technology to measure the size, concentration, and zeta potential of particles in liquid suspensions. It provides detailed information about the properties of particles ranging from 10 nanometers to 1 micrometer in size.
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58 protocols using zetaview pmx 120
1
Nanoparticle Tracking Analysis of EVs
2
Nanoparticle Tracking Analysis of EVs
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The size distribution and concentration of EVs were analysed by NTA on a ZetaView PMX-120 instrument (Particle Metrix, Inning am Ammersee, Germany). All samples were diluted in NaCl-HEPES to a final volume of 1 mL. The manufacturer’s default software settings were selected for EV analysis. For each measurement, two cycles were performed by scanning 11 cell positions. The following camera settings were used: Shutter: 100 (sEVs), 150 (mEVs); Sensitivity: 85 (sEVs), 75 (mEVs); Framrate: 60 (sEVs), 7.5 (mEVs); cell temperature: 25 °C. The videos were analysed with minimum brightness of 20, a minimum area of 5 and a maximum area of 1000 by ZetaView Analyze software 8.05.10.
3
Nanoparticle Size and Concentration Analysis
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The NTA experiment was conducted using Zetaview PMX 120 (Particle Metrix, Meerbusch, Germany). A 1 ml sample was diluted with 1 × PBS and added to a new cell. Three cycles were performed by scanning 11 cell positions each and capturing 60 frames per position. The outliers of those measurements were removed. After capture, the videos were analyzed using ZetaView Software 8.04.02 with the following analysis parameters: maximum particle size, 1000; minimum particle size, 5; minimum particle brightness, 20. The average and median sizes and the concentration of the particles were calculated based on the data of the optimized positions.
4
Exosome Size and Distribution Analysis
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Isolated exosomes diluted in PBS were gently mixed, and the mixture was inserted into the ZetaView PMX120 instrument (Particle Metrix GmbH) at 23°C through a 1-ml syringe. ZetaView 8.02.28 software (Particle Metrix GmbH) was utilized to analyze the number and the size distribution of the exosomes. Each sample was measured three times and the average value was calculated.
5
Characterization of scFv(AM1)-P-BAP Hybrid Polyplexes
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Multiparameter nanoparticle tracking analysis was performed using the ZetaView® PMX120 (Particle Metrix, Inning am Ammersee, Germany) according to the manufacturer’s instructions to determine the particle quantities, zeta potential, and particle size of scFv(AM1)-P-BAP-hybrid polyplexes with MC. Hybrid polyplexes were prepared as described above. Data were analyzed using the manufacturer’s software (ZetaView 8.05.05).
6
Exosome Purification from BMDM Cultures
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Exosome purification was performed as previously described (Cui et al., 2019 (link)). Briefly, the bone-marrow-derived macrophages (BMDMs) were cultured for 72 h. The debris and dead cells in the cultured medium were removed by centrifugation at 1,000 × g for 10 min at 4°C. After filtration through a 2-μm filter, the medium was subjected to ultracentrifugation at 100,000 × g for 6 h at 4°C. The pellets were washed with PBS and then centrifuged at 100,000 × g for 20 min at 4°C. An exosome-containing pellet was collected and resuspended in PBS. The particle size and concentration of exosome were determined using network address translation (NTA) with ZetaView PMX 120 (Particle Metrix).
7
Nanoparticle Characterization via ZetaView
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The samples were diluted in ultrapure water and the numbers of particles were measured using ZetaView® PMX120 (Particle Metrix, Meerbusch, Germany). Measurements were done once at all 11 positions and the video quality was set to medium. Lower detection limit was provided by the manufacturer. The dilution factor varied between 50 and 500 depending on the sample. The measured volume was 1 ml and the measurement time 10 s. The chamber temperature was set to 22 °C, and the sensitivity of the camera to 85. Data were analysed using the ZetaView® analysis software version 8.05.12 with a minimum area of 10, a maximum area of 1000 and a minimum brightness of 30.
8
Nanoparticle Size Analysis via NTA
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The NTA experiment was carried out using Zetaview PMX 120 (Particle Metrix, Meerbusch, Germany). Briefly, a 2 mL sample was diluted with 1 × PBS and added into a new cell. The instrument measured 11 different positions of the cell, and the outliers of those measurements were removed. The average and median sizes and the concentration of the particles were calculated based on the data of the optimized positions. In this study, the optimal measurement condition settings were: temperature at 25 °C, sensitivity at 70 °C, shutter speed at 70 s, and conductivity at 15,000 μs/cm.
9
Nanoparticle Size Analysis of Extracellular Vesicles
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The samples were diluted between 1:10,000 (v/v) and 1:1000 in high-purity water (MilliQ-System, 18.2 MΩ cm resistivity at 25 °C, Millipore, Merck, Darmstadt, Germany) or 40 mm ammonium acetate, pH 8.4 for measurement, depending on the origin of EV samples. All measurements were performed with a ZetaView PMX120 instrument (ParticleMetrix, Meerbusch, Germany) at 22 °C. The instrument determines the hydrodynamic particle diameter along 11 laser-light scattering measurement positions. The expected minimum brightness of the particles was set to auto, the particle size range to 5–200 nm, the shutter to 100, and the minimum tracelength and frame rate each to 15. The obtained data were analyzed with the corresponding instrument software (ZetaView 8.05.05 SP2, ParticleMetrix) to calculate median (X50), standard deviation, and size distribution, resulting in an average concentration of 1011–1012 particles/mL for EV samples and 108–1010 particles/mL for SEC samples.
10
Nanoparticle Characterization via NTA and SEC
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For NTA, the samples were diluted 1:10,000 (v:v) and 1:50,000 (v:v), for SEC samples 1:1000 (v:v) in high-purity water (18.2 MΩcm resistivity at 25 °C, MilliQ-System, Merck, Darmstadt, Germany), or 40 mM ammonium acetate solution, pH 8.4 and immediately measured after dilution. The measurements were performed with a Zetaview PMX120 (ParticleMetrix, Meerbusch, Germany) at 22 °C. Each measurement consists of 11 scattering measurement positions. The minimum brightness of the expected particles was set to auto, the particle size range was set to 5–200 nm, the shutter was set to 100, the minimum tracelength was set to 15, and the frame rate was set to 15 (arbitrary units, each). On average, the concentration of EV samples was 1011–1012 particles per milliliter, while SEC samples only contained around 108–109 particles per milliliter. The obtained data was analyzed with the corresponding instrument software (ZetaView 8.05.05 SP2) to calculate mean, standard deviation, and size distribution.
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