Mouse brain ECs were cultured as described above. Exosomes were isolated using ExoQuick-TC (Exosome Precipitation Solution kit, System Biosciences) following previously described protocol12 (link), and suspended in PBS. EC-Exo are from non-diabetic condition. Briefly, media was isolated from a 100% confluent T75 tissue culture flask (containing ~5–6 million cells) containing 10 ml of media with exosome free FBS. This media was centrifuged and filtered via a 0.22 μm syringe filter before adding ExoQuickTC (2 ml for 10 ml media). Exosome number was counted using the IZON qNano device (Izon). To decrease the heterogeneity of Exo treatments, we tightly controlled the cell culture conditions, such as passage, density, and culture time. Liposomes are synthetic versions of exosomes mimicking the exosomal lipid layer and were generated using thin-film hydration technique as previously described13 (link).
Protocol detail
Exosome Isolation from Mouse Brain Endothelial Cells
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Corresponding Organization :
Other organizations : Henry Ford Hospital, Oakland University
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Variable analysis
independent variables
- Exosome treatment
- Liposome treatment
- Exosome number
- Cell culture conditions (passage, density, culture time)
- Exosome isolation protocol (centrifugation, filtration, ExoQuick-TC treatment)
- Liposome generation protocol (thin-film hydration technique)
- Positive control: Non-diabetic condition exosomes (EC-Exo)
- Negative control: Not specified
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