Visualization of Exosome Release and Uptake During Cell Migration Using the Live Imaging Reporter pHluorin_M153R-CD63

Methods Mol Biol. 2023;2608:83-96. doi: 10.1007/978-1-0716-2887-4_6.


Exosome secretion and uptake regulate cell migration through autocrine and paracrine mechanisms. Monitoring exosome secretion and uptake during cell migration is critical for investigation of these mechanisms. Exosomes can be visualized by direct labeling with fluorescent dyes or by tagging intrinsic markers with fluorescent proteins for live imaging. Due to several limitations of fluorescent dye-labeled exosomes, we created two bright genetically encoded reporters of exosome secretion, pHluorin_M153R-CD63 and pHluorin_M153R-CD63-mScarlet. Here, we describe how to visualize secretion and uptake of exosomes labeled with these pH-sensitive and pH-insensitive fluorescent protein-tagged exosomal markers during cell migration using time-lapse fluorescent microscopy.

Keywords: Cell migration; Exosome labeling; Exosome secretion; Exosome uptake; Live cell imaging; Multivesicular body trafficking; Time-lapse fluorescent microscopy; pHluorin_M153R-CD63; pHluorin_M153R-CD63-mScarlet.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biological Transport
  • Cell Movement
  • Exosomes* / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism


  • PHluorin
  • Green Fluorescent Proteins