Exosome isolation from plasma problems

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jseibert
jseibert's picture
Exosome isolation from plasma problems

 Hello All,

I just started an exosome isolation (from plasma) protocol (by invitrogen) this week and I am having trouble pelleting the cell debris. I first thaw all my samples on ice, then stick them in the centrifuge at 2000 x g for 20 min @ RT. After this step, there is somewhat of a pellet at the bottom (I place all of my microcentrifuge cap hinges on the outside so even if I can’t see the pellet, I still treat it as though there is a pellet). As I am aspirating the supernatant (using either a p1000, p200, or p100) I seem to pick up cell debris right around where the meniscus is located (the debris also comes from the same side of the tube that the pellet is located on). I transferred the supernatant anyway to a new tube because I will be centrifuging again at 10,000 x g for 20 min. However, the same problem came up, so I spun it down again at 15,000 x g for 20 min. It still did not fix the problem.

Is there anything I can do to not pick up any cell debris? I would like to refrain from using a smaller pipeteman (less suction) because this would take more time.
Any ideas would be greatly appreciated!
Jake

Ketil Winther P...
Ketil Winther Pedersen's picture
 Hi, 

 Hi, 

feel free to check our work on exosome isolation from plasma combining size exclusion chromatography and Dynabeads CD9 isolation at: https://www.researchgate.net/profile/Ketil_Pedersen

kind regards
ketil

Ketil Winther P...
Ketil Winther Pedersen's picture
 Hi, we have just published a

 Hi, we have just published a paper demonstrating how to optimize direct capture of exosomes using Dynabeads - without any pre-enrichment such as by ultracentrifugation. (Pedersen et al 2015 Translational Biomedicine). The paper can be downloaded at http://bit.ly/1bX8X7R together with other scientific information about exosomes. 

kind regards
ketil