Hello,
I've been doing some exosome/vesicle extractions. I've done some before with PC3 cells, resulting in a tiny white pellet. However, this is the first time doing this with LNCaP.
Following concentrating the conditioned media (growth media: phenol red containing RPMI-1640- serum free), I removed cellular debris and dead cells, then ultracentrifuged at 100,000xg.
The issue is the resulting pellet is red. Bright red. Probably coming from the phenol red. But is it normal for exosome pellets to be coloured?
Thanks in advance.
Most likely your Exosomes were not completely sealed when you started working on it, so they have encapsulated the phenol red. You should make sure that at the start the buffer is PBS or something with the same amount of salt (150mM NaCl) which keeps the exosomes sealed. Please let me know which buffer are you using.
Judging from what you say, you're following Thery's 2006 protocol with a concentration step beforehand to increase yield. I've never performed the concentration with UC, but phenol red never got caught by the cutoff filter when I did SEC.
I've never seen colored pellets before, even working with pigmented cells, and all my media contains phenol red.
I do know bright red pellets are generated when creating exosome-depleted media. Those pellets are massive, brilliantly red/orange, and nigh impossible to dislodge. You're absolutely certain your RPMI is serum free? Nobody added FBS/FCS/etc and didn't say anything? Lots of things can get mixed around in a lab, especially the bigger ones and even 1% generates a sizeable pellet.
Thank you for your reply.
I am 100% sure these are serum free medias. In fact it was a brand new bottle of RPMI-1640 from Gibco, opened the day of my first EV extraction preparation where this pellet was observed.
In the days following this extraction my cells was presented with an overgrowth of fungus (tiny cells with filamentous growth). So, I was thinking that maybe my pellets had actually contained some of that.
Domenico, I'm not sure by what your referring to when you say starting buffer, given that the extraction is done in growth media.
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