EVs were prepared from the supernatant of primary astrocytes and by differential centrifugations as previously described (Hu et al., 2012 (link); Hu et al., 2013 (link); Hu et al., 2018 (link)). Briefly, primary astrocytes were treated with HIV Tat protein for 24 hours. Then, conditioned media were harvested, centrifuged at 1,000 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 100,000g 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).
Protocol detail
Isolation and Characterization of Astrocyte-Derived Extracellular Vesicles
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Assay
Astrocytes
Cell
Centrifugations
Conditioned media
Exosome
Filtration
Hiv tat protein
Protein
Tsg101
Ultracentrifugation
Western blot
Corresponding Organization : University of Nebraska Medical Center
Other organizations : Creighton University
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Variable analysis
independent variables
- Treatment of primary astrocytes with HIV Tat protein
- Protein content of extracellular vesicles (EVs)
- Presence of exosome markers TSG101 and CD63 in EVs
- Quantification of EVs using nanoparticle tracking analysis
- Preparation of EVs from the supernatant of primary astrocytes using differential centrifugations as previously described
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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