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Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection

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    Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection
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    † Department of Chemistry and Biochemistry, Jackson State University, Jackson, Mississippi 39217, United States
    ‡ Cancer Institute, University of Mississippi Medical Center, Jackson, Mississippi 39150, United States
    § Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39150, United States
    ∥ Department of Biology, Tougaloo College, Tougaloo, Mississippi 39174, United States
    *E-mail: [email protected]. Phone: +1-601-9793491.
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    Analytical Chemistry

    Cite this: Anal. Chem. 2016, 88, 21, 10390–10394
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    https://doi.org/10.1021/acs.analchem.6b03348
    Published September 30, 2016
    Copyright © 2016 American Chemical Society
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    Abstract

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    Chemical analysis of small extracellular vesicles (sEVs) circulating in body fluids holds potentials in noninvasive diagnosis of diseases and evaluation of therapeutic treatments. However, quantification of sEVs remains a challenge due to lacking of cost-effective analytical protocols. Herein we report a facile method based on size exclusion chromatography with fluorescence detection (SEC-FD) for sEVs quantification. After removal of cells and cell debris, a 0.50 mL sample (e.g., cell culture medium) is incubated with CM-Dil dye to fluorescently label sEVs. The incubation solution is then separated on a SEC column packed with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553 nm/Em570 nm by using a fluorometer equipped with a 50-μL flow through cuvette. Separation efficiency of the proposed SEC-FD method was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate. Liposomes were eluted out in less than 6 min, about 10 min before albumin-FITC. A separation repeatability (RSD in retention time) of 1.4% (n = 5) was obtained for liposomes. In analysis of cell culture media, linear calibration curves based on SEC-FD peak height versus sEVs concentration were obtained with r2 value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (n = 5). The detection limit was estimated to be 2.9 × 107 exosome particles/mL. The proposed assay was applied to the first study of sEVs secretion from TK6 cells cultured in serum-free medium for a culturing period from 1 to 48 h.

    Copyright © 2016 American Chemical Society

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03348.

    • DSL measurements of the liposome preparation; identification of SEC-FD peaks by DSL, TEM, and protein assay; and calibration curve for exosome quantification (PDF)

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    Analytical Chemistry

    Cite this: Anal. Chem. 2016, 88, 21, 10390–10394
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.analchem.6b03348
    Published September 30, 2016
    Copyright © 2016 American Chemical Society
    Request reuse permissions

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