Display options
Cite
Görgens A, Bremer M, Ferrer-Tur R, et al. Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material. J Extracell Vesicles. 2019;8(1):1587567doi: 10.1080/20013078.2019.1587567.
Görgens, A., Bremer, M., Ferrer-Tur, R., Murke, F., Tertel, T., Horn, P. A., Thalmann, S., Welsh, J. A., Probst, C., Guerin, C., Boulanger, C. M., Jones, J. C., Hanenberg, H., Erdbrügger, U., Lannigan, J., Ricklefs, F. L., El-Andaloussi, S., & Giebel, B. (2019). Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material. Journal of extracellular vesicles, 8(1), 1587567. https://doi.org/10.1080/20013078.2019.1587567
Görgens, André, et al. "Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material." Journal of extracellular vesicles vol. 8,1 (2019): 1587567. doi: https://doi.org/10.1080/20013078.2019.1587567
Görgens A, Bremer M, Ferrer-Tur R, Murke F, Tertel T, Horn PA, Thalmann S, Welsh JA, Probst C, Guerin C, Boulanger CM, Jones JC, Hanenberg H, Erdbrügger U, Lannigan J, Ricklefs FL, El-Andaloussi S, Giebel B. Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material. J Extracell Vesicles. 2019 Mar 21;8(1):1587567. doi: 10.1080/20013078.2019.1587567. eCollection 2019. PMID: 30949308; PMCID: PMC6442110.
Copy Download .nbib Format: NLM
Share it on

J Extracell Vesicles. 2019 Mar 21;8(1):1587567. doi: 10.1080/20013078.2019.1587567. eCollection 2019.

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material.

Journal of extracellular vesicles

André Görgens, Michel Bremer, Rita Ferrer-Tur, Florian Murke, Tobias Tertel, Peter A Horn, Sebastian Thalmann, Joshua A Welsh, Christine Probst, Coralié Guerin, Chantal M Boulanger, Jennifer C Jones, Helmut Hanenberg, Uta Erdbrügger, Joanne Lannigan, Franz L Ricklefs, Samir El-Andaloussi, Bernd Giebel

Affiliations

  1. Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
  2. Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
  3. Evox Therapeutics Limited, Oxford, UK.
  4. Luminex B.V., 's-Hertogenbosch, Netherlands.
  5. Translational Nanobiology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
  6. Amnis/Luminex, Seattle, WA, USA.
  7. Paris Descartes University, Paris, France.
  8. Institut Curie, cytometry core, PSL University, Paris, France.
  9. INSERM, U970, Paris Cardiovascular Research Center-PARCC, Paris, France.
  10. Department of Pediatrics III, University Children's Hospital Essen, University Duisburg-Essen, Essen, Germany.
  11. Department of Medicine, Nephrology Division, University of Virginia, Charlottesville, VA, USA.
  12. Flow Cytometry Core, University of Virginia School of Medicine, Charlottesville, VA, USA.
  13. Department of Neurological Surgery, University Medical Center Hamburg Eppendorf, Hamburg, Germany.
  14. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

PMID: 30949308 PMCID: PMC6442110 DOI: 10.1080/20013078.2019.1587567

Abstract

Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.

Keywords: CD63; Extracellular vesicles; exosomes; flow cytometry; imaging flow cytometry; microvesicles; reference material; standardisation; submicron particle analysis

References

  1. Hum Gene Ther. 2003 Apr 10;14(6):509-19 - PubMed
  2. Cytometry B Clin Cytom. 2004 Jan;57(1):1-6 - PubMed
  3. Cytometry A. 2004 Jun;59(2):237-45 - PubMed
  4. Anal Biochem. 2007 May 15;364(2):180-92 - PubMed
  5. Clin Lab Med. 2007 Sep;27(3):653-70, viii - PubMed
  6. Exp Cell Res. 2009 May 15;315(9):1584-92 - PubMed
  7. J Thromb Haemost. 2009 Jan;7(1):190-7 - PubMed
  8. ACS Nano. 2010 Apr 27;4(4):1921-6 - PubMed
  9. J Thromb Haemost. 2010 Nov;8(11):2571-4 - PubMed
  10. J Thromb Haemost. 2010 Dec;8(12):2596-607 - PubMed
  11. Blood. 2011 Jan 27;117(4):e39-48 - PubMed
  12. Semin Thromb Hemost. 2010 Nov;36(8):807-18 - PubMed
  13. J Virol. 2011 Feb;85(4):1452-63 - PubMed
  14. Methods Mol Biol. 2011;699:67-84 - PubMed
  15. J Thromb Haemost. 2011 Jun;9(6):1216-24 - PubMed
  16. Nat Commun. 2011;2:282 - PubMed
  17. Nanomedicine. 2011 Dec;7(6):780-8 - PubMed
  18. Colloids Surf B Biointerfaces. 2011 Oct 1;87(1):146-50 - PubMed
  19. J Thromb Haemost. 2011 Aug;9(8):1679-81; author reply 1681-2 - PubMed
  20. J Thromb Haemost. 2011 Aug;9(8):1676-8; author reply 1681-2 - PubMed
  21. Nanomedicine. 2012 Jul;8(5):712-20 - PubMed
  22. J Thromb Haemost. 2012 May;10(5):919-30 - PubMed
  23. Nat Protoc. 2012 Jun 14;7(7):1311-26 - PubMed
  24. Cytometry A. 2013 Feb;83(2):242-50 - PubMed
  25. PLoS One. 2012;7(11):e49726 - PubMed
  26. J Cell Biol. 2013 Feb 18;200(4):373-83 - PubMed
  27. J Extracell Vesicles. 2013 Feb 15;2:null - PubMed
  28. J Extracell Vesicles. 2013 May 27;2:null - PubMed
  29. Cytotherapy. 2014 Feb;16(2):170-80 - PubMed
  30. Leukemia. 2014 Apr;28(4):970-3 - PubMed
  31. J Thromb Haemost. 2014 May;12(5):614-27 - PubMed
  32. J Thromb Haemost. 2014 Jul;12(7):1182-92 - PubMed
  33. Cytometry A. 2014 Sep;85(9):756-70 - PubMed
  34. Sci Rep. 2014 Jun 10;4:5237 - PubMed
  35. J Immunol Methods. 2014 Sep;411:55-65 - PubMed
  36. Biochem J. 2015 Jan 1;465(1):103-14 - PubMed
  37. Stem Cell Reports. 2014 Dec 9;3(6):1058-72 - PubMed
  38. Nanomedicine. 2015 May;11(4):879-83 - PubMed
  39. Cytometry A. 2016 Feb;89(2):135-47 - PubMed
  40. Methods. 2015 Oct 1;87:3-10 - PubMed
  41. J Extracell Vesicles. 2015 Mar 31;4:25530 - PubMed
  42. Cytometry A. 2015 Nov;87(11):1052-63 - PubMed
  43. J Extracell Vesicles. 2015 Apr 20;4:26316 - PubMed
  44. Cytometry A. 2016 Feb;89(2):148-58 - PubMed
  45. PLoS One. 2015 May 15;10(5):e0127209 - PubMed
  46. J Extracell Vesicles. 2015 May 14;4:27066 - PubMed
  47. Curr Protoc Cytom. 2015 Jul 01;73:13.14.1-16 - PubMed
  48. Stem Cells Transl Med. 2015 Oct;4(10):1131-43 - PubMed
  49. Transl Res. 2015 Dec;166(6):733-9 - PubMed
  50. Cytometry A. 2016 Feb;89(2):196-206 - PubMed
  51. J Extracell Vesicles. 2015 Dec 23;4:29509 - PubMed
  52. J Extracell Vesicles. 2015 Dec 31;4:30087 - PubMed
  53. Cell Cycle. 2016;15(4):540-5 - PubMed
  54. Proc Natl Acad Sci U S A. 2016 Feb 23;113(8):E968-77 - PubMed
  55. J Extracell Vesicles. 2016 Feb 19;5:29975 - PubMed
  56. ACS Nano. 2016 Apr 26;10(4):3886-99 - PubMed
  57. Stem Cells Transl Med. 2016 Jun;5(6):754-63 - PubMed
  58. J Extracell Vesicles. 2016 Jun 24;5:29254 - PubMed
  59. Cytometry A. 2016 Jul;89(7):663-72 - PubMed
  60. Front Immunol. 2016 Jul 26;7:282 - PubMed
  61. Eur J Pharm Sci. 2017 Feb 15;98:4-16 - PubMed
  62. Methods. 2017 Jan 1;112:55-67 - PubMed
  63. Brain Behav Immun. 2017 Feb;60:220-232 - PubMed
  64. Ann Transl Med. 2017 Mar;5(6):150 - PubMed
  65. Circ Res. 2017 May 12;120(10):1632-1648 - PubMed
  66. Sci Rep. 2017 May 12;7(1):1878 - PubMed
  67. Sci Rep. 2017 Sep 14;7(1):11561 - PubMed
  68. Nanomedicine. 2018 Apr;14(3):801-810 - PubMed
  69. Chem Rev. 2018 Feb 28;118(4):1917-1950 - PubMed
  70. Front Immunol. 2018 Apr 30;9:738 - PubMed
  71. J Thromb Haemost. 2018 Jun 7;:null - PubMed
  72. Front Immunol. 2018 Jun 13;9:1326 - PubMed
  73. Front Immunol. 2018 Jul 06;9:1583 - PubMed
  74. J Extracell Vesicles. 2018 Oct 17;7(1):1528109 - PubMed
  75. J Extracell Vesicles. 2018 Nov 23;7(1):1535750 - PubMed
  76. J Biol Chem. 1998 Aug 7;273(32):20121-7 - PubMed
  77. J Virol. 1998 Nov;72(11):8873-83 - PubMed

Publication Types