Abstract
Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,microarrays and can beutilized in functional in vitro/in vivo studies.
Highlights
Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, microRNAs, metabolites, and lipids
We confirmed the presence of EV-protein in gluteal- and abdominal-derived EVs by immunoblotting for tumor susceptibility gene 101 (TSG101) and found that abdominal and gluteal adipocyte cell pellets and abdominal and gluteal adipocyte-derived EVs were positive for TSG101, whereas as sham-control media, which had not been in contact with cells was negative (Figure 1E)
We demonstrate a protocol for the isolation of gluteal and abdominal adipocyte-derived EVs from cell culture supernatants and determine their size and concentration by NTA7, 14, 15
Summary
Metabolic disturbances, including obesity, insulin resistance, and perturbations in extracellular glucose, oxygen, and inflammation can alter the size and concentration of EVs and their cargo. Ultracentrifugation remains a popular method of isolation for EVs because it is accessible and requires little prior specialist knowledge. Other methods such as precipitation, size exclusion chromatography, and immunoaffinity capture using tetraspanins enable EV isolation from a range of biofluids, including plasma, serum, urine, and conditioned cell culture media. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EVprotein tumor susceptibility gene 101 (TSG101) in our human adipocyte derived EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNAsequencing, microarray, and can be utilized in functional in vitro/in vivo studies. Add 50 mL of the warmed BSA solution to each tube and mix well by repeated vortexing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
