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. 2020 Sep;88(1):e110.
doi: 10.1002/cpcb.110.

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

Christina Coughlan  1 , Kimberley D Bruce  2 , Olivier Burgy  3 , Timothy D Boyd  4 , Cole R Michel  5 , Josselyn E Garcia-Perez  6 , Vanesa Adame  4 , Paige Anton  7 , Brianne M Bettcher  8 , Heidi J Chial  1 , Melanie Königshoff  9 , Elena W Y Hsieh  10   10 , Michael Graner  11 , Huntington Potter  1
Affiliations

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

Christina Coughlan et al. Curr Protoc Cell Biol. 2020 Sep.

Abstract

Exosomes are 50- to 150-nm-diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation- and column-based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek® ) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream® ). High-performance liquid chromatography followed by nano-flow liquid chromatography-mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5-fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Pre-analytic fluid collection and processing Basic Protocol 2: Exosome isolation by ultracentrifugation Alternate Protocol 1: Exosome isolation by precipitation Basic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysis Alternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometry Basic Protocol 4: Downstream analysis of exosomes using high-performance liquid chromatography Basic Protocol 5: Downstream analysis of the exosome proteome using nano-flow liquid chromatography-mass spectrometry.

Keywords: exosome; isolation; lipoproteins; mass spectrometry; ultracentrifugation.

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Figures

Figure 1.
Figure 1.. Schematic of the workflow used to isolate and analyze exosomes from plasma.
Schematic of the workflow used to isolate and analyze exosomes from plasma. Initial blood sample were processed to generate plasma samples (Basic Protocol 1), which were used to isolate exosomes using either ultracentrifugation (Basic Protocol 2 ) or precipitation (Alternate Protocol 1). Isolated exosomes were then characterized at a single cell resolution by nanoparticle tracking analysis (Basic Protocol 3) or using flow cytometry and imaging flow cytometry (Alternate Protocol 2). Deep profiling was also carried out by high resolution liquid chromatography (Basic Protocol 4) and nano-flow liquid chromatography mass spectrometry (Basic Protocol 5). Overall expected time is indicated for each described protocol. Abbreviation: SN : supernatant.
Figure 2.
Figure 2.. Plasma exosome quantification with the Exo-Flow-ONE staining kit.
A PRE exosome samples (Alternate Protocol 1) were stained with Exo-Flow ONE. a) Unstained exosome sample, showing no spectral signature for the detectors. b) Exosome sample stained with Exo-Flow-ONE garnet far red showing a spectral signature in channel R2 consistent with the excitation/emission 628/643 nm. Data from a representative PRE exosome sample is shown.
Figure 3.
Figure 3.. Quantification of plasma exosomes using the Exo-Flow-ONE staining kit.
PRE exosome samples (Alternate Protocol 1) were serially diluted as shown above, and each diluted sample was stained with Exo-Flow ONE. Exosome count (read as event count on the flow cytometer) is plotted against the corresponding dilution factor demonstrating a linear correlation between the count and the dilution factor. Data from a representative PRE exosome sample is shown.
Figure 4.
Figure 4.. Immunophenotyping of plasma exosomes using ImageStream.
PRE exosome samples (Alternate Protocol 1) were stained with fluorescently labeled anti-CD9 (FITC), anit-CD81 (PE), and anti-CD63 (APC) antibodies and analyzed via ImageStream. Exosomes that express CD9 (FITC), CD81 (PE), and CD63 (APC) (left to right) are shown. Each row depicts an individual exosome. Data from a representative PRE exosome sample is shown.
Figure 5.
Figure 5.
Schematic for high speed pump (analytical pump), capillary pump (loading pump), multisampler (injector), trap column, and analytical column connections and flow paths in both analytical and loading positions in the multiple column thermostat UHPLC switching valve.
See this image and copyright information in PMC

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