ECVs were analyzed by NanoSight model NS300 (Malvern Instruments Company, Nanosight, Malvern, United Kingdom) as described earlier [22 (link)]. Briefly, ECV pellet samples obtained in the process of ECV isolation were resuspended in 100 μl 4% paraformaldehyde-PBS and further diluted 100 fold in PBS for analysis. Three 90 s videos were recorded for each sample. The obtained data were analyzed using Nanosight NTA 2.3 Analytical Software (Malvern Instruments Company) with the detection threshold optimized for each sample and screen gain at 10 to track the maximal number of particles with minimal background. Most of isolated ECVs carry fluorescent Aβ (1–40) cargo. In addition, Aβ is associated with ECVs of different size (see Additional file 1 : Figure S1).
Nanosight nta 2.3 analytical software
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The Nanosight NTA 2.3 Analytical Software is a tool designed for the characterization of nanoparticles. It utilizes nanoparticle tracking analysis (NTA) technology to measure the size distribution and concentration of particles in liquid samples.
Automatically generated - may contain errors
5 protocols using nanosight nta 2.3 analytical software
1
Characterization of Amyloid-Beta Carrying Extracellular Vesicles
2
Exosome Size and Concentration Analysis
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The concentration and size distribution of the isolated exosomes was analyzed by Nanosight NS300 system (Malvern Instruments Company, Nanosight, and Malvern, United Kingdom). Briefly, exosome preparations were homogenized by vortexing followed by serial dilution of 1:1000 in sterile Phosphate saline buffer and analyzed by NanoSight NS300. Each sample analysis was conducted for 90 minutes. Data was analyzed by Nanosight NTA 2.3 Analytical Software (Malvern Instruments Company, Nanosight, and Malvern, United Kingdom) with the detection threshold optimized for each sample and screen gain at 10 to track as many particles as possible with minimal background. Polystyrene latex standards were analyzed to validate the operation of the instrumentation and a blank 0.2 μm-filtered 1x PBS was also run as a negative control. At least three analysis were done for each individual sample.
3
Nanoparticle Characterization of Extracellular Vesicles
4
Exosome Size and Concentration Analysis
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The concentration and size distribution of the isolated exosomes was analyzed by NanoSight NS300 system (Malvern Instruments Company, Nanosight, and Malvern, United Kingdom). Briefly, EV preparations were homogenized by vortexing followed by serial dilution of 1:1,000 in sterile Phosphate buffer saline (PBS) and analyzed with the NanoSight NS300. Each sample analysis was conducted for 90 min. Data were analyzed by Nanosight NTA 2.3 Analytical Software (Malvern Instruments Company, Nanosight, and Malvern, United Kingdom) with the detection threshold optimized for each sample and screen gain at 10 to track as many particles as possible with minimal background. Polystyrene latex standards were analyzed to validate the operation of the instrumentation and a blank 0.2 μm-filtered 1x PBS was also run as a negative control. At least three analysis were done for each individual sample.
5
Nanoparticle Tracking Analysis of hCMEC/D3 Extracellular Vesicles
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EVs isolated from the supernatant of hCMEC/D3 cells were characterized and analyzed by nanoparticle tracking analysis (NTA) on the Nanosight NS300 (Malvern Instruments Company, Malvern, UK). Optimal detection settings were acquired with the screen gain set to 11 and a detection threshold level of 5 using Nanosight NTA 2.3 Analytical Software (Malvern Instruments Company). This allowed us to track the EV particles in suspension with minimal background [Supplementary Video 1 and Supplementary Video 2 ]. EV pellets were diluted 100-fold in sterile-filtered PBS and five 15-s frame captures were recorded for each sample. An average of three batches from each group was used to determine the total number of vesicles. EV-Aβ40 vesicle number was normalized to the total average number of EV-PBS for all treatments.
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