Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection.
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 r2value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (n = 5). The detection limit was estimated to be 2.9 × 107exosome 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.
Department of Chemistry and Biochemistry, Jackson State University , Jackson, Mississippi 39217, United States.
Rui
R
Xu
Department of Chemistry and Biochemistry, Jackson State University , Jackson, Mississippi 39217, United States.
Austin
A
Fitts
Department of Chemistry and Biochemistry, Jackson State University , Jackson, Mississippi 39217, United States.
Xiangtang
X
Li
Cancer Institute, University of Mississippi Medical Center , Jackson, Mississippi 39150, United States.
Joseph
J
Fernandes
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.
Radhika
R
Pochampally
Department of Biology, Tougaloo College , Tougaloo, Mississippi 39174, United States.
Jinghe
J
Mao
Department of Chemistry and Biochemistry, Jackson State University , Jackson, Mississippi 39217, United States.
Yi-Ming
YM
Liu
D002232:Carbocyanines / Q000032:analysis*; D002460:Cell Line; D002850:Chromatography, Gel / Q000379:methods*; D000067128:Extracellular Vesicles / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D010316:Particle Size
C434385:3,3'-dioactadecyl-5,5'-di(4-sulfophenyl)oxacarbocyanine; D002232:Carbocyanines; D005456:Fluorescent Dyes; D008081:Liposomes
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J Immunol. 2007 Aug 15;179(4):2235-41
17675484
Cell. 2016 Mar 10;164(6):1226-1232
26967288
Curr Protoc Cell Biol. 2006 Apr;Chapter 3:Unit 3.22
18228490
PLoS One. 2014 Oct 13;9(10):e109631
25310591
Nature. 2015 Jul 9;523(7559):161-2
26106856
J Control Release. 2015 Feb 28;200:87-96
25555362
Science. 2014 Dec 19;346(6216):1459-60
25525233
PLoS One. 2015 Dec 21;10(12):e0145686
26690353
J Extracell Vesicles. 2012 Nov 21;1:
24009887
Cancer Biomark. 2010-2011;8(4-5):281-91
22045359
J Transl Med. 2011 Jun 08;9:86
21651777
J Histochem Cytochem. 2015 Mar;63(3):181-9
25473095
Front Immunol. 2014 Oct 21;5:525
25374572
J Thromb Haemost. 2014 Jul;12(7):1182-92
24818656
J Extracell Vesicles. 2015 Jul 06;4:27378
26154623
Sci Rep. 2014 Jun 10;4:5237
24913598
J Extracell Vesicles. 2014 Mar 25;3:
24678386
J Transl Med. 2014 Aug 08;12:204
25104112
J Extracell Vesicles. 2016 Mar 14;5:31053
26979463