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EM and Exosome

Thanks Ketil,

Q1: I followed the protocol of total exosome isolation (from cell culture media) using 1 ml of serume free-cell media collected 24 hrs. do you think it will be enongh exosomse to do EM.


Q2: I have used Exosome resuspension buffer to resusoendind the exosomes pellet after spin down at 10,000g for 1hrs and then use it  to do a western ananlysis using anti-CD9 (Cat No: WB10626D)1 in 500 dilution. Exosome is unable to be detected.
related to an answer for: EM and exsomes
asked Mar 17, 2015 in ExosomesTalk by anniewang
any success?

1 Answer

hi,
Q1) in principle I will assume that will be more than enough. However, it will of course depend on your cells and how "efficient" they are in exosome production. it is also important to pay attention to the exosome harvest and collect the exosomes at the right time. which time point is correct will depend on the cell type. One way of checking this is actually to use e.g. Dynabeads CD9 and pull out a small fraction of exosomes each day (without any pre-enrichment) during cell culture and stain for CD9 and perform analysis by flow cyt. this will give you an idea of exosome release and which would be the best period of performing exosome harvest and produce exosomes suitable for negative stain, immuno labelling and TEM analysis.
Q2) If you are not able to detect exosomes using CD9 this could be explained either by too little starting material, exosomes not expressing CD9 or the use of reducing conditions during sample prep for WB - as we have tested this WB clone for several years on many different samples and know that it works very well on exosomes. would have to look at your protocol in more detail to see if there are something to change. you may send it to me
kind regards

ketil
answered Mar 19, 2015 by ketil.pedersen
edited Sep 7, 2015 by ketil.pedersen
Thanks you very much. i have got it working now.
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