This might be a simple question, but I can’t find any paper to explain it accurately.
I will be soon doing FACS to isolate neurons from C. elegans, and after talking to the FACS technician of my university, he suggests NOT to use FBS, that PBS is enough.
I was wondering if anyone could explain what could be better and why?
Really appreciated!
Vicky
Hi Vicky,
It’s been a while since I’ve been around those doing c. elegans cell culture, but my grad lab was one of the pioneers in determining how to best culture embryonic cells and sort them. The key factor was determining that the osmolality of the solution needed to be optimized for C. elegans cells vs mammalian cells. PBS is not optimized for worm cells, and thus you won’t get good results from sorting cells in them - they will be very unhappy. Our institution at the time was happy to flush out the PBS and use our solution of choice (I think egg buffer), but many FACS cores don’t want to do that since their bread and butter is sorting mammalian cells.
I’m not privy to the latest/greatest methods in this, but if FBS is the suggested sorting medium then that’s what I would do. I know that Yun Zhang in the Chalfie group, which was the first published sorting method, did use FBS. Our group didn’t have the resources the Chalfie group had, and FBS is expensive, so we were lucky that our core was ok with flushing out the system.
I hope that helps! But please, if anyone has more updated information, please point out anything I’ve gotten incorrect.
Best,
Steve
Hi Vicky,
We have done quite a bit of cell sorting, and as Steve said, the most important factor for cell viability is the osmolarity of the buffer, but I would lean towards using egg buffer rather than PBS since it is optimized for worms. In our hands, we get the best results sorting in egg buffer conditioned to mOsm 340 ± 5. If your downstream application is cell culture, I recommend collecting cells into L-15 (also conditioned to mOsm 340 ±5). See the following papers/wormbook chapter for details.
Good luck!
Aaron
https://www.nature.com/articles/nprot.2007.143
https://elifesciences.org/articles/30201
http://www.wormbook.org/chapters/www_cellculture/cellculture.html