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Is there anyone who are isolating exosomes from ESC culture medium?

Is there anyone who are isolating exosomes from ESC culture medium? what your culture condition for isolating exosomes? what kind of medium? how long,24,48 or 72 hours? and how much medium(mL) is sufficent for isolating exosome? look forward to replies. thank you~
asked Dec 8, 2013 in ExosomesTalk by anonymous
My email is [email protected], we can keep in touch to have some discussion.

1 Answer

we did not try this particular cell line, but for all that we tested - number of secreted exosomes and isolation protocols are very similar. When growing cells, use chemically defined media or exosome-depleted FBS; exosomes can be collected at 24h; can use 1-5 mL media to isolate plenty of exosomes- enough for several qRT-PCR or Westerns; sequencing and some other types of downstream analysis require more material, so use 30 ml cell media.
answered Dec 9, 2013 by Vlassosv
thank you very much for your answers. Is the pellet visible isolated from just 1-5mL medium? because I have tried 4mL medium, but I didn't get visible pellet. or the pellet is just invisible?
when you isolate exosomes from cell media- the pellet is invisible in most cases, unless you use a large volume (eg 30 ml). because the concentration of exosomes is rather low, and the preparation is very clean. With serum, plasma and other body fluids the pellet is typically visible if you process over 100 uL sample.
OK, thank you so much.
If I wanna do a western analysis, how should I process the pellet after 10000g, 1h(Total Exosome Isolation Reagent (from cell culture media)). should I air dry the pellet?  what kind of buffer should I use, PBS, lysis buffer, or directly add loading buffer? how much buffer should I add(if the start cell culture medium is 10mL)?
I have tried several times but none of the exo marker(CD9, Alix, TSG101) were detected. and when I get the pellet from large volume medium, I found the pellet is hard to dissolve.
Hi Johnny you can check our paper: The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo. Jeoffrey Schageman, Emily  Zeringer, Mu Li, Tim Barta, Kristi Lea, Jian Gu, Susan Magdaleno, Robert Setterquist, and Alexander V. Vlassov. Hindawi Publishing Corporation, BioMed Research International. Volume 2013, Article ID 253957, 15 pageshttp://dx.doi.org/10.1155/2013/253957. and another one. both papers are free access and can be found at www.lifetechnologies.com/exosomes
you can add the loading buffer (standard, whatever you use for other westerns) directly to the exosomes pellet (no need to air dry it), heat and move FW with western gel. Can load exosome equivalent of 1 ml cell media per gel lane, and if the signal is strong- later on reduce the amount. it depends on the protein, your Ab, etc. If you have problems with westerns- it could be several things, so better start with the positive control, eg highly abundant protein GAPDH, and not exosomes but cells. if everything looks fine- go from there
There are several typical reasons why Western Blotting analysis does not work:
1.    Not enough sample volume added. Exosomes can contain a fairly low amount of protein cargo, so for an initial experiment we recommend adding as much of the sample as possible
2.    Antibodies are not optimal. We suggest testing antibodies (e.g. anti-CD63 or other exosomal marker) from 2-3 manufacturers, carefully checking what concentration is recommended. Also, they should ideally be used fresh, and need to be stored properly.
3.    Depending on the exosomal surface marker, certain gel conditions might be more optimal for the target antibody (e.g. reducing/nonreducing and denaturing/nondenaturing). We suggest checking with the manufacturer and exosome community about which WB conditions are recommended for the specific marker you are targeting and the specific antibody you are using.
4.    General western techniques. Westerns can be tricky so we recommend the use of a positive control for initial testing to make sure the entire workflow is functioning as it should.  Any protein or antibody can be used as long as they meet the conditions you need (e.g. denaturing vs. non-denaturing). In addition, when picking the protein, try to steer clear of those that are present at very high or very low concentrations in your sample to prevent overloading the blot or total absence of signal.
Thank u very much. your information is very detailed.
I'll try. hope to solve my problem.
Thanks
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