In article <klosen-180194163226 at 130.104.130.2> klosen at bani.ucl.ac.be (Klosen
Paul) writes:
>We are having trouble to differentiate PC12 cells with NGF. So far we have
>tested four different batches of PC12 cells and NGf from 3 different
>sources. Has anyone encountered similar problems and how to solve them ????
>>--
>Paul Klosen
>Cell Biology Lab., Catholic University of Louvain
>klosen at bani.ucl.ac.be>Make sure you plate your PC12 cells on collagen or poly-lysine/laminin- coated
dishes and not just on tissue culture plastic. The NGF will make the cells
less adherent to the substratum and they will fall off or not grow out
processes unless you coat the plastic. They will also grow processes out more
readily and rapidly in serum-free medium (eg., with N-2 supplements ala
Bottenstein/Sato.)
Many people have become very sloppy about maintaining their PC12 cell lines.
They should be passaged before they overgrow the dish. What happens after many
passages in cell culture is that you start selecting for cells that no longer
respond to NGF because the non-responders divide more rapidly than the
responsive cells. A "real" PC12 cell should divide once every 2-3 days. If
your cells look especially flat on tissue culture plastic, they probably don't
respond to NGF anymore. If this happens, then you must subclone your cell line
and select for the subclones that have retained NGF-responsiveness.
Rae Nishi
OHSU
Portland OR USA
