Recently, there was a problem with my qpcr results. I want to try more methods of extracting EV from the cell medium. At present, I am using Invitrogen's kit(Cat4478359) to extract exosomes. Can you give me more suggestions? Thank you very much!
Dear Na Tang,
You can use this article:
Konoshenko MY, Lekchnov EA, Vlassov AV, Laktionov PP. Isolation of extracellular vesicles: general methodologies and latest trends. BioMed research international. 2018 Oct;2018.
I hope that it would be useful.
Dear Na Tang,
1st Step: Please centrifuge your cell culture media at 315 g for 12 min at 4 degree Celsius to pellet dead cells and debris.
2nd Step: The remaining supernatant can then be centrifuged at 2000 g for 10 min at 4 degree Celsius to pellet apoptotic bodies.
3rd Step: The remaining supernatant can next be centrifuged at 10,000 g for 30 min at 4 degree Celsius to pellet multi-vesicular bodies.
4th Step: The remaining supernatant can then be centrifuged once more at 100,000 g for 70 min at 4 degree Celsius to pellet exosomes.
Finally, the pellet of apoptotic bodies/multi-vesicular bodies/exosomes can be re-suspended in PBS and processed for down-stream applications.
Hope this helps.
Best - Pradyumna
Dear Na Tang,
You can isolate exosomes and extract RNA for analysis. It takes two steps; see publication:Article Novel HIV-1 miRNAs stimulate TNFα release in human macrophag...
Step 1: Isolate exosomes from cell culture conditioned medium using ExoQuick-TC reagent (System Biosciences, Mountain View, California).
Step 2: Total RNA is extracted from exosomes using a SeraMir Exosome RNA Purification Column kit (System Biosciences, cat. no. RA808A-1).
Cheers,
Mark Bernard
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