EVs were prepared from the supernatant of primary astrocytes and by differential centrifugations as previously described (Hu et al. 2012 (link), 2013 (link), 2018 (link)). Briefly, primary astrocytes were treated with HIV Tat protein for 24 h. Then, conditioned media were harvested, centrifuged at 1000 g for 10 min to eliminate cells, and again spun at 10,000 g for 30 min, followed by filtration through 0.22 μm filter to remove cell debris. EVs were pelleted by ultracentrifugation (Beckman Ti70 rotor, Brea, CA, USA) at 100,000 g for 70 min. EVs were assessed for their protein content using BCA Protein Assay Kit (Pierce, Rockford, IL, USA). TSG101 and CD63 were detected by western blot as exosome markers. EVs were further quantified using the ZetaView nanoparticle tracking analysis system (Particle Metrix, Mebane NC).
Zetaview nanoparticle tracking analysis system
Manufactured by Particle Metrix
Sourced in Germany
The ZetaView nanoparticle tracking analysis system is a laboratory instrument designed to measure the size, concentration, and surface charge (zeta potential) of nanoparticles in liquid suspensions. It utilizes nanoparticle tracking analysis (NTA) technology to provide real-time visualization and quantification of individual nanoparticles.
Automatically generated - may contain errors
3 protocols using zetaview nanoparticle tracking analysis system
1
Isolation and Characterization of Astrocyte-Derived Extracellular Vesicles
2
Isolation and Characterization of Astrocyte-Derived Extracellular Vesicles
3
Characterizing Extracellular Vesicle Size
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Hydrodynamic particle size of crude EV samples in PBS were measured in triplicate using a Zetasizer Nano-ZS (Malvern Instruments, Malvern Hills, United Kingdom). Particle concentration and size distribution were determined using a ZetaView nanoparticle tracking analysis system (Particle Metrix, Germany) and ZetaView software (version 8.05.11 SP1). Hundred nanometer polystyrene standard particles were used for calibration measurements. For video acquisition: sensitivity=85, shutter speed=100, acquisition=30 frames per second. Each sample was measured at 11 different positions, with 2 cycles of readings at each position. After automated analysis and removal of any outliers from the 11 positions, the size (diameter in nm) and the concentration (particles/mL) were calculated.
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