Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids
- SourceAvailable from: Chider Chen
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- "Cells were cultured in exosome-depleted medium (complete medium depleted of FBS-derived exosomes by overnight centrifugation at 100,000 g) for 48 hr. Exosomes from culture supernatants were isolated by differential centrifugation, as described in the literature (Thery et al., 2006), at 300 g for 10 min, 2,000 g for 10 min, 10,000 g for 30 min, and 100,000 g for 70 min, followed by washing with PBS and purification by centrifugation at 100,000 g for 70 min. In each exosome preparation, the concentration of total proteins was quantified using the Pierce BCA Protein Assay (Thermo). "
ABSTRACT: Mesenchymal stem cell transplantation (MSCT) has been used to treat human diseases, but the detailed mechanisms underlying its success are not fully un- derstood. Here we show that MSCT rescues bone marrow MSC (BMMSC) function and ameliorates osteopenia in Fas-deficient-MRL/lpr mice. Mecha- nistically, we show that Fas deficiency causes failure of miR-29b release, thereby elevating intracel- lular miR-29b levels, and downregulates DNA meth- yltransferase 1 (Dnmt1) expression in MRL/lpr BMMSCs. This results in hypomethylation of the Notch1 promoter and activation of Notch signaling, in turn leading to impaired osteogenic differentiation. Furthermore, we show that exosomes, secreted due to MSCT, transfer Fas to recipient MRL/lpr BMMSCs to reduce intracellular levels of miR-29b, which re- sults in recovery of Dnmt1-mediated Notch1 pro- moter hypomethylation and thereby improves MRL/ lpr BMMSC function. Collectively our findings un- ravel the means by which MSCT rescues MRL/lpr BMMSC function through reuse of donor exosome- provided Fas to regulate the miR-29b/Dnmt1/Notch epigenetic cascade.Cell Metabolism 08/2015; DOI:10.1016/j.cmet.2015.08.018
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- "can be analyzed if bound to beads coated with antibodies against surface antigens, such as MHC class II. Subsequent labeling by fluorophore-conjugated antibodies enables semiquantitative evaluation by flow cytometry (Théry et al. 2006). Flow cytometry analysis of EVs from body fluids, however, can be disturbed by immune complexes that have similar biophysical properties as EVs (György et al. 2011). "
ABSTRACT: The release of extracellular vesicles (EVs), including exosomes and microvesicles, is a phenomenon shared by many cell types as a means of communicating with other cells and also potentially removing cell contents. The cargo of EVs includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. EVs released into the extracellular space can enter body fluids and potentially reach distant tissues. Once taken up by neighboring and/or distal cells, EVs can transfer functional cargo that may alter the status of recipient cells, thereby contributing to both physiological and pathological processes. In this article, we will focus on EV composition, mechanisms of uptake, and their biological effects on recipient cells. We will also discuss established and recently developed methods used to study EVs, including isolation, quantification, labeling and imaging protocols, as well as RNA analysis.BioScience 07/2015; 65(783). DOI:10.1093/biosci/biv084
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- "Exosomes are typically derived from the inward budding of multivesicular bodies (MVBs) and are of interest because they serve as highly efficient export vehicles (Thé ry et al., 2002). Immunofluorescent staining of EUCVs revealed colocalization of several proteins frequently found in exosomes, including the ESCRT proteins Alix and Tsg101, as well as the tetraspanin CD63 (Thery et al., 2006) (Figure 2A). To more quantitatively confirm the presence of exosome markers on EUCVs, the bacteria were first covalently linked to magnetic beads before exposure to BECs (Lö nnbro et al., 2008), and EUCV-encased bacteria were isolated from cell-free medium on a magnetic rack. "
ABSTRACT: Vertebrate cells have evolved elaborate cell-autonomous defense programs to monitor subcellular compartments for infection and to evoke counter-responses. These programs are activated by pathogen-associated pattern molecules and by various strategies intracellular pathogens employ to alter cellular microenvironments. Here, we show that, when uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because of their capacity to neutralize lysosomal pH. This change is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to lysosomes. TRPML3 activation then spontaneously initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. These studies reveal a cellular default system for lysosome homeostasis that has been co-opted by the autonomous defense program to clear recalcitrant pathogens. Copyright © 2015 Elsevier Inc. All rights reserved.Cell 05/2015; 161(6). DOI:10.1016/j.cell.2015.05.009
Questions & Answers about this publication
- Any suggestions on the approaches to study exosome?
Specifically how can I isolate exosome from condition medium and ascites, how to break down exosome and study the composition of membrane (such as lipid, protein) as well as contents (miRNA, DNA). Thank you very much.
The "classic" paper on exosome isolation and characterization is the one from Théry et al. (Isolation and characterization of exosomes from cell culture supernatants and biological fluids), of which I've added the link below to request a full text.
I have recently published another study on isolation methods and the resulting purity myself, where you can also find some protocols for protein and RNA analysis. This paper is open access (second link). Based on these results I would advise to stay away from commercial kits if you can. For the highest purity, go for an OptiPrep or sucrose density gradient.Following
- Did anyone isolate exosomes for microRNA extraction from MSC or any other cell lines?
If you did,what is the best method to isolate exosomes for microRNA extraction? How much culture media need to collect or how many cells need to culture?
Remember to use completely vesicle-free media, preferably serum-free, as you will otherwise isolate bovine RNA that for the most part cannot be distinguished from RNA of your species of interest. If your goal is to isolate exosomes, you will not obtain reasonably pure exosomes by doing only a slow first spin to remove cells. At a minimum, sequential centrifugations at higher g are needed to remove cell debris, apoptotic bodies, and larger vesicles. Showing that a putative exosomal marker is present is also not the same thing as demonstrating that co-purifying complexes are absent!Following