The size distribution and concentration of exosomes were analyzed by NTA, according to the manufacturer's instructions. Briefly, exosomes were resuspended and diluted at 1:200 in PBS, and the particle concentration of exosomes was assessed using a NanoSight NS500 Instrument (Malvern Instruments, Ltd.).
Nanosight ns500 instrument
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The NanoSight NS500 instrument is a nanoparticle characterization system that utilizes Nanoparticle Tracking Analysis (NTA) technology to measure the size, concentration, and particle size distribution of a wide range of nanomaterials and nanoparticles suspended in liquids. The instrument captures video of particles moving under Brownian motion and analyzes the trajectory of each particle to determine its hydrodynamic size and concentration.
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Lab products found in correlation
Whatman, by Cytiva (2 mentions)
Em109 tem, by Zeiss (2 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by Lipoid (1 mentions)
1 2 distearoyl sn glycero 3 phosphoethanolamine n methoxy polyethylene glycol 2000 mpeg2000 dspe, by Lipoid (1 mentions)
Bortezomib, by LC Laboratories (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Nta software, by Malvern Panalytical (1 mentions)
Igg1 sc 34665, by Santa Cruz Biotechnology (1 mentions)
Siteclick qdot 605 antibody conjugation kit, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Polystyrene latex microbeads, by Thermo Fisher Scientific (1 mentions)
Nta 3.0 analytical software, by Malvern Panalytical (1 mentions)
Exoeasy maxi kit, by Qiagen (1 mentions)
Zetasizer nano zs instrument, by Malvern Panalytical (1 mentions)
Jsm 7600f field emission scanning electron microscope fesem, by JEOL (1 mentions)
Buffer xe, by Qiagen (1 mentions)
Nta 3, by Malvern Panalytical (1 mentions)
Nanosight nta software version 3, by Malvern Panalytical (1 mentions)
Zetasizer nano s instrument, by Malvern Panalytical (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
20 protocols using nanosight ns500 instrument
1
Nanoparticle Tracking Analysis of Exosomes
2
Liposomal Bortezomib Formulation and Characterization
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1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG2000-DSPE) were obtained from Lipoid GmbH, Germany. Cholesterol and Whatman® Anodisc inorganic membranes were obtained from Sigma Aldrich, Germany. Bortezomib was purchased from LC Laboratories, Canada. High-pressure extruder from Lipex, Northern Lipids, Canada was used for liposome resizing. NanoSight NS500 instrument from Malvern Panalytical, UK was used for particle size determination. Ultra-high Performance Liquid Chromatography (UPLC) from Waters Corporation, USA was used for determination of Bortezomib concentrations.
3
Exosome Characterization via Qdot Labeling
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Qdots (Qdot nanocrystals) were conjugated to anti-CD63 (45 ), which is mainly associated with membranes of intracellular vesicles and, thus, is widely used as a marker of small EVs such as exosomes, PLAP, or immunoglobulin (Ig) G1 isotype control antibody (IgG1 sc-34665, Santa Cruz Biotechnology) (45 ) with a SiteClick Qdot 605 Antibody Conjugation Kit (Life Technologies), executed according to the manufacturer’s instructions as previously described (42 (link),46 ). Exosomes were diluted in PBS and incubated with FcR blocking reagent (10 µL, 10 minutes at 4°C) (MACS Miltenyi Biotec), followed by incubation with anti-CD63-Qdot605, PLAP–Qdot605, or IgG1-Qdot605 (10 µL, 1:100) for 30 minutes in the dark at room temperature. Samples (ie, [1] exosomes alone, [2] exosomes + IgG1-Qdot605, [3] exosomes + anti-CD63-Qdot605 and exosomes + anti-PLAP-Qdot605, [4] background controls, [5] FcR blocking reagent + IgG1-Qdot605, [6] FcR blocking reagent + anti-CD63-Qdot605, and [7] FcR blocking reagent + anti-PLAP-Qdot605) were then diluted to 500 µL with PBS and analyzed using the NanoSight NS500 instrument and NTA software (Malvern Panalytical Ltd., Malvern, UK). In fluorescence mode (ie, camera level 9, shutter speed 11.25 ms and slider gain 250), five 60-second videos were captured for each sample and analyzed.
4
Nanoparticle Characterization from PBMC
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Effects and molecular constituents of EVs present in PBMCsec were reported previously [30 (link), 32 (link)]. Nanoparticles were isolated and reconstituted in phosphate-buffered saline (PBS) as described previously [30 (link)] (Fig. 1 b, c). Qualitative and quantitative assessments of nanoparticles were performed by the Molecular Neuro-Oncology, Department of Pediatrics and Adolescent Medicine and Institute of Neurology, Medical University of Vienna (Vienna, Austria), using the NanoSight NS500 instrument (Malvern Instruments, Malvern, UK). For each measurement, 500 μL of the diluted sample was loaded by the automatic pump control into the NanoSight system. The repetition and duration of captures were set manually (5 captures with 30 s each) and used for all measurements. The instrument was calibrated using 100-nm-particle reference controls provided by the instrument manufacturer.
5
Nanoparticle Size Determination Protocol
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Quantification and size determination of BMEVs were assessed by using a NanoSight NS500 instrument (Malvern). The instrument was set up to operate at room temperature. Three videos were recorded for each specimen, and the results were analyzed with NTA software.
6
Exosome Quantification and Sizing
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Quantification and size determination of exosomes was performed using a NanoSight NS500 instrument (Malvern Panalytical, Malvern, UK) by the Laboratory of Nanostructures of the Polish Academy of Science (Warsaw, Poland). NanoSight NTA 2.3 (build 0025) software (Malvern Panalytical) was used for the visualization and analysis of nanoparticles. Samples of exosomes derived from cell cultures were diluted 1:10 in PBS for optimal concentration before examination with the NTA system. The instrument operating temperature was set to 25 °C, and each sample was tested in 5 replicates.
7
Characterizing Exosome Size Distribution
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Isolated exosomes were examined under a Zeiss EM109 TEM (Carl Zeiss) as detailed previously (Wu et al. 2015 (link)). The size distribution of isolated exosomes was measured using a Nano Sight NS500 instrument (Malvern Instruments Ltd, Malvern, UK) (Ruf et al. 1989 (link)).
8
Nanoparticle Tracking Analysis of Extracellular Vesicles
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Nanoparticle tracking analysis (NTA) was performed using a NanoSight NS500 Instrument (Malvern Instruments Inc., Westborough, MA) equipped with a 532 nm laser and integrated automated fluidics. Five 60-second videos were recorded of each sample with camera level set at 14 and detection threshold set at 6. Temperature was set at 23 °C and monitored throughout the measurements. Videos recorded for each sample were analyzed with NTA software version 3.0 to determine the concentration and size of measured particles with corresponding standard error. For analysis, auto settings were used for blur, minimum track length and minimum expected particle size. The NanoSight system was calibrated with polystyrene latex microbeads of 50, 100, and 200 nm (Thermo Scientific Inc.) prior to analysis. PBS− (Gibco #10010-023) was used as diluent and to avoid contaminating particles a fresh bottle was opened for each analysis session. At least three separate EV preparations for each condition were analyzed.
9
Nanoparticle Size Analysis via NTA
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Nanoparticle tracking analysis (NTA) was performed using a Nanosight NS500 instrument (Malvern Panalytical Ltd., Malvern, UK). The particles were 1000× diluted in PBS solution. The temperature during the measurement was 22 °C based on the NTA Temperature program. NTA recorded 30 s videos of each measurement of the sample by a CCD camera.
10
Nanoparticle Characterization using NanoSight
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