Exosome Isolation: Cyclical Electrical Field Flow Fractionation in Low-Ionic-Strength Fluids.
ANALYTICAL CHEMISTRY(2018)
摘要
The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, lx phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at similar to 1.3 V AC. A375 melanoma exosome recovery was approximately 70 - 80% after each buffer substitution, and recovery was independent of whether the sample was substituted into lx PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using lx PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from similar to--1.6 to -0.1 mu m cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from similar to--5.5 to -0.1 mu m cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.
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