Abstract
Over the last 10 years, the constant progression in exosome (Exo)-related studies highlighted the importance of these cell-derived nano-sized vesicles in cell biology and pathophysiology. Functional studies on Exo uptake and intracellular trafficking require accurate quantification to assess sufficient and/or necessary Exo particles quantum able to elicit measurable effects on target cells. We used commercially available BODIPY® fatty acid analogues to label a primary melanoma cell line (Me501) that highly and spontaneously secrete nanovesicles. Upon addition to cell culture, BODIPY fatty acids are rapidly incorporated into major phospholipid classes ultimately producing fluorescent Exo as direct result of biogenesis. Our metabolic labeling protocol produced bright fluorescent Exo that can be examined and quantified with conventional non-customized flow cytometry (FC) instruments by exploiting their fluorescent emission rather than light-scattering detection. Furthermore, our methodology permits the measurement of single Exo-associated fluorescence transfer to cells making quantitative the correlation between Exo uptake and activation of cellular processes. Thus the protocol presented here appears as an appropriate tool to who wants to investigate mechanisms of Exo functions in that it allows for direct and rapid characterization and quantification of fluorescent Exo number, intensity, size, and eventually evaluation of their kinetic of uptake/secretion in target cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cocucci E, Meldolesi J (2015) Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends Cell Biol 25(6):364–372. doi:10.1016/j.tcb.2015.01.004
Felicetti F, Parolini I, Bottero L et al (2009) Caveolin-1 tumor-promoting role in human melanoma. Int J Cancer 125(7):1514–1522. doi:10.1002/ijc.24451
Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9(8):581–593. doi:10.1038/nri2567
Maas SLN, de Vrij J, van der Vlist EJ et al (2015) Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics. J Control Release 200:87–96. doi:10.1016/j.jconrel.2014.12.041
Nolte-'t Hoen EN, van der Vlist EJ, Aalberts M et al (2012) Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles. Nanomedicine 8(5):712–720. doi:10.1016/j.nano.2011.09.006
van der Vlist EJ, Nolte-'t Hoen EN, Stoorvogel W et al (2012) Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protoc 7(7):1311–1326. doi:10.1038/nprot.2012.065
Kormelink TG, Arkesteijn GJ, Nauwelaers FA et al (2015) Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry. Cytometry A 89:135–147. doi:10.1002/cyto.a.22644
Pospichalova V, Svoboda J, Dave Z et al (2015) Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer. J Extracell Vesicles 4:25530. doi:10.3402/jev.v4.25530
Parolini I, Federici C, Raggi C et al (2009) Microenvironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem 284(49):34211–34222. doi:10.1074/jbc.M109.041152
Thery C, Amigorena S, Raposo G et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 30:3.22.21–3.22.29. doi:10.1002/0471143030.cb0322s30
Tauro BJ, Greening DW, Mathias RA et al (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56(2):293–304. doi:10.1016/j.ymeth.2012.01.002
Thumser AE, Storch J (2007) Characterization of a BODIPY-labeled fluorescent fatty acid analogue. Binding to fatty acid-binding proteins, intracellular localization, and metabolism. Mol Cell Biochem 299(1–2):67–73. doi:10.1007/s11010-005-9041-2
Wang H, Wei E, Quiroga AD et al (2010) Altered lipid droplet dynamics in hepatocytes lacking triacylglycerol hydrolase expression. Mol Biol Cell 21(12):1991–2000. doi:10.1091/mbc.E09-05-0364
Record M, Carayon K, Poirot M et al (2014) Exosomes as new vesicular lipid transporters involved in cell-cell communication and various pathophysiologies. Biochim Biophys Acta 1841(1):108–120. doi:10.1016/j.bbalip.2013.10.004
Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1):497–509
Kobayashi T, Stang E, Fang KS et al (1998) A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392(6672):193–197. doi:10.1038/32440
Bhattacharya AA, Grüne T, Curry S (2000) Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin1. J Mol Biol 303(5):721–732. doi:10.1006/jmbi.2000.4158
Schröder M, Schäfer R, Friedl P (1997) Spectrophotometric determination of iodixanol in subcellular fractions of mammalian cells. Anal Biochem 244(1):174–176. doi:10.1006/abio.1996.9861
Acknowledgment
This work was supported by the Italian Ministry of Health (grant RF-2011-02347300). We thank Mario Falchi for his expert assistance with confocal imaging.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Coscia, C. et al. (2016). Generation, Quantification, and Tracing of Metabolically Labeled Fluorescent Exosomes. In: Federico, M. (eds) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Methods in Molecular Biology, vol 1448. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3753-0_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3753-0_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3751-6
Online ISBN: 978-1-4939-3753-0
eBook Packages: Springer Protocols