To visualize and quantify the EV content of unfractionated vitreous samples, Nanoparticle Tracking Analysis (NTA) was performed as described previously [14 (link)], where tracked particles are presumed to represent EVs based on prior analysis [14 (link)]. This step was performed prior to abundant protein depletion. Briefly, vitreous samples were removed from storage at − 80 °C, thawed on ice, and centrifuged at 2000 g for 30 min at 4 °C. The supernatants were diluted to 1 ml [1:50 to 1:1000] with particle-free water. Each prepared sample was loaded by syringe pump into the NanoSight NS300 (Malvern Instruments Ltd, Malvern, Worcestershire, UK) set to scatter mode, and five 60-s videos were generated at 24.98 frames per second. The size distribution and concentration of particles were calculated using NanoSight software version 3.2 (Malvern Instruments Ltd, Malvern, Worcestershire, UK). All samples in the current data set were run by the same individual. The raw NTA data were processed using Microsoft Excel (Redmond, WA, USA). Individual tracings from a single vitreous sample were averaged, and the averaged data for all samples within a single phenotype were again averaged to yield a final graph. Particle abundance values of zero were replaced with blank cells to reflect their interpretation as undetected rather than truly zero.
Nanosight software version 3
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The NanoSight software version 3.2 is a core analytical tool used for the characterization of nanoparticles. It provides detailed information about the size, size distribution, and concentration of particles in liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using nanosight software version 3
1
Quantifying Vitreous Extracellular Vesicles
2
Nanoparticle size analysis of vitreous samples
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Vitreous samples were removed from storage at −80°C, thawed on ice, and centrifuged at 2000g for 30 minutes at 4°C. The supernatants were diluted to 1 mL (1:50 to 1:1000) with particle-free water. Each sample was loaded by syringe pump into the NanoSight NS300 (Malvern Instruments Ltd, Malvern, Worcestershire, UK) set in scatter mode, and five 60-second videos were generated at 24.98 frames/sec. The size distribution and concentration of particles were calculated and images were acquired using NanoSight software version 3.2 (Malvern Instruments Ltd). The same individual ran all samples in the current data set on the same day.
The raw NanoSight data were analyzed using Microsoft Excel algorithms (Redmond, WA). The algorithm used identified all ‘peaks' across the entire size spectrum (1–1000 nm parsed into 1-nm bins). For this study, peaks were operationally defined as a nanometer value, n, whose vesicle concentration value was greater than both n − 1 and n + 1. Only peaks accounting for >5% of the total vesicle population were considered for further analysis. For each such peak, the total vesicle abundance of the peak was calculated by adding the vesicle concentrations of nanometer values ranging from n − 5 to n + 5. This range was chosen because the standard deviation across all NanoSight runs was approximately 11 nm.
The raw NanoSight data were analyzed using Microsoft Excel algorithms (Redmond, WA). The algorithm used identified all ‘peaks' across the entire size spectrum (1–1000 nm parsed into 1-nm bins). For this study, peaks were operationally defined as a nanometer value, n, whose vesicle concentration value was greater than both n − 1 and n + 1. Only peaks accounting for >5% of the total vesicle population were considered for further analysis. For each such peak, the total vesicle abundance of the peak was calculated by adding the vesicle concentrations of nanometer values ranging from n − 5 to n + 5. This range was chosen because the standard deviation across all NanoSight runs was approximately 11 nm.
3
Quantification of Extracellular Vesicles
4
Exosomes Quantification Using NanoSight
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Exosomes quantification was performed using a NanoSight NS300 (Malvern Instruments Ltd, Malvern) previously (56 ). Briefly, exosome samples were diluted in 1 ml of particle free-water, and each sample was loaded by syringe pump into the NanoSight NS300 (Malvern Instruments Ltd, Malvern) set in scatter mode, and five 60-s videos were generated at 24.98 frames/sec. The size distribution and concentration of particles were calculated, and images were acquired using NanoSight software, version 3.2 (Malvern Instruments Ltd).
5
Nanoparticle Size Analysis of EV Suspensions
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EV suspensions were diluted to 1 mL (1:50 to 1:1000) with particle-free water. Each sample was loaded by syringe pump into the NanoSight NS300 (Malvern Instruments Ltd, Malvern, Worcestershire, UK) set in scatter mode, and five 60-second videos were generated. The size distribution and concentration of particles were analyzed and images were acquired using NanoSight software version 3.2 (Malvern Instruments Ltd).
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