Fluoresecently label exosome cargo
to monitor cellular interaction & delivery
- Label endogenous exosome RNAs red
- Monitor exoRNA delivery
- Label internal exosome proteins green
- Track exosome protein delivery
Fluorescently-label exosome RNAs and proteins to monitor trafficking
Exosomes are 60 - 180 nm membrane vesicles secreted by most cell types in vivo and in vitro and contain distinct subsets of RNAs and proteins depending upon the cell type from which they are secreted, making them useful for biomarker discovery and functional characterization. These nano-sized shuttles transport signaling RNAs and proteins to other cells.
The Exo-Glow kits allow you to fluorescently-label isolated exosome RNAs and proteins to track cellular interaction and cargo delivery. You can fluorescently label exosome internal RNA Red or internal exosome proteins Green for tracking. Isolated exosomes are incubated with either the 10x Exo-Red (for RNAs) or 10x Exo-Green (for proteins) labeling reagent. The Exo-Red stain is based on an Acridine Orange (AO) chemistry. AO is membrane permeable and fluorescently-labels single-stranded RNAs inside of exosomes. These Red exoRNAs can then be monitored for delivery into target cells via the exosomes using fluorescent microscopy. The Exo-Green stain is based on Carboxyfluorescein succinimidyl diacetate ester (CFSE) chemistry. CFSE is also membrane permeable. When CFSE enters exosomes it is hydrolyzed by exosome esterases (Savina, A., 2002) known to be within exosomes that remove the diacetate residues. This activates the CFSE to fluoresce green and is then it is coupled to the amino ends of exosome proteins. This approach allows you to track internal exosome protein transfer into target cells using fluorescent microscopy.
How the exosome labels work
The Exo-Red exosome label is based on Acridine Orange chemistry and is a nucleic acid selective fluorescent cationic dye. It is cell-permeable, and interacts with DNA by intercalation and RNA by electrostatic attractions. When bound to DNA, Exo-Red is very similar spectrally to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm (green). When Exo-Red associates with RNA, the excitation maximum shifts to 460 nm (blue) and the emission maximum shifts to 650 nm (red). As the exoRNA is delivered into the target cell, you may see punctate structures glowing red corresponding to RNA/protein complexes within the cells. Over time (about 6 hours later), transfer of the fluorescent color to green may occur. This simply indicates that the exoRNA has been delivered into the cell and the Exo-Red label has dissociated from the exoRNA after delivery, allowing Exo-Red to now bind to DNA inside of the cell and then fluoresce green.
The Exo-Green stain is based on Carboxyfluorescein succinimidyl diacetate ester (CFSE) chemistry. CFSE is membrane permeable. When CFSE enters exosomes it is hydrolyzed by esterases known to be within exosomes that remove the diacetate residues. This activates the CFSE to fluoresce green and is then coupled to the amino ends of proteins, making them fluoresce green. Exo-Green has an excitation maximum at 494 nm and an emission maximum at 521 nm (green).