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How to best differentiate between intra- and extra-cellular membrane structures?

How can I make sure that the exosomes I purify are not intracellular structures (pieces of ER etc.)?The only option I see is to enrich cell medium by ultracentrifugation or 'exosome isolation' reagents (e.g. Invitrogen, SBI) and then select for cell surface markers. However, these markers should be NOT expressed on intracellular membranes, but on ALL extracellular membranes - any ideas? Or is there other options than antibodies?
asked Apr 15, 2013 in ExosomesTalk by Franziska

1 Answer

Hi Franziska. great question. when you are isolating exosomes/extracellular vesicles from eg cell media, or body fluid such as blood- the first step is gentle centrifugation, in order to get rid of cells and cell debris. Whatever remains in supernatant is exosomes, microvesicles, proteins. Then you can recover exosomes using the Total exosome isolation reagents, and- if required- obtain ultra pure population of CD63 positive exosomes with Dynabeads-CD63. You are right that its not trivial (at least at the moment) to capture the specific populations using some antibodies (or other affinity reagents)- as there are no absolutely specific markers for exosomes or other vesicles. Its not a problem for many projects, unless you suspect your cells are "leaky" and release some components into the media. CD63, CD81, CD9, Alix, Annexin, TSG are not strictly specific exosome markers. we need to come up with a panel of positive and negative markers for each type of vesicles, and sub-populations as well. Might take several years to do that. I am looking forward to great discussions at the ISEV meeting in Boston this week.
answered Apr 16, 2013 by Vlassosv