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Current Protocols in Cell Biology

Institut Curie, Paris, France.
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 05/2006; Chapter 3(Unit 3):Unit 3.22. DOI: 10.1002/0471143030.cb0322s30
Source: PubMed

ABSTRACT

Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.

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    • "Transfections were carried out in T-75 flasks. The cell-free supernatants from four T-75 flasks (40 ml of medium) were combined and subjected to a series of centrifugation steps to isolate exosomes as previously described (Thery et al. 2006). Briefly, the conditioned medium was first clarified by centrifugation at 300×g for 10 min, and the supernatant was centrifuged at 2000×g for 10 min. "
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    ABSTRACT: In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aβ) and Aβ peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND.
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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). "
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    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.
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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). "
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Questions & Answers about this publication

  • Jan Van Deun added an answer in Exosomes:
    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.

    Jan Van Deun

    Hi Yilin,

    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.

    + 1 more attachment

  • Kenneth W Witwer added an answer in Exosomes:
    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?

    Thanks!

    Kenneth W Witwer

    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!

    • [Show abstract] [Hide abstract]
      ABSTRACT: Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.
      No preview · Article · May 2006 · Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]

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