Dear Colleagues,
For Exosomes/Extracellular vesicles (EVs) labelling by PKH26 stain, and studying their cellular uptake.
Is there anyone who tried to mix conditioned medium with PKH26 to stain Exosomes/Extracellular vesicles (EVs) before isolation with targeted filtration?
I am using the kit "101 PureExo" for targeted filtration and isolation of EVs.
Hello Islam Saadeldin
PKH26 dye is not specific to EVs. It will stain fragments of membrane or any lipid bilayer for that matter. So, it will often label EVs non-specifically as other cellular components also get labelled thereby giving false positive signals. Lipophilic dyes like PKH are not reliable EV labelling agents unless and until you have an entirely pure population of EVs devoid of cellular components.
Any specific reason to mix conditioned medium with PKH 26?
Best.
Dear Malcolm Nobre
Thank you for the feedback. I will use a kit that isolates 95% of pure EVs (101 Bio, PureExo kit). Would you please give me your ideas about how to study EVs uptake by cells?
Hi,
It won't be an appropriate way of labeling exosomes. As explained by Malcolm Nobre by doing so you will label other cells components. For Pkh26 Exo labeling first, dilute Exo and Pkh26 dye in the diluent C available in the kit, combine them and keep for some minutes at RT, stop the reaction by adding serum and PBS wash by ultracentrifuge to remove unbound dye, and you are good to use them for cellular uptake studies.
Good luck.
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