U52608 at UICVM.UIC.EDU wrote:
: Hi, Dear Netter,
: I am using isolated rat or mice hepatocyte nuclei to perform nuclei uptake stud
: ies. Therefore my nuclei preparation have to be pure, intact, and ALIVE (FUNCTI
: ONAL). Does anybody know how to get such perfect FUNCTIONAL nuclei? And how am
: I going to test if my nuclei preparation is functional or not? For how long can
: the isolated nuclei maintain functional? Any trick to preserve nuclei intact?
: I've followed the methods described in PNAS 86:453, 1989, to isolate nuclei and
: did similar calcium uptake studies. But my results are not consistent. Also the
: magnitude of Ca active uptake is far less than the authors described on that
: paper. I am quite frustrated. The authors are in Dept of Toxicology, Karolinska
: Institutet, Stockholm, Sweden. I've tried Gopher to search their e-mail addres
: s, but I failed. Does anyone know how to get their e-mail address, if there is?
: Any suggestion is highly appreciated. Thanks a lot.
Getting nuclei out with intact membranes (which is what you'll have to do,
I think) is difficult. Most of the procedures for isolating nuclei use
detergent lysis of the plasmalemma and take advantage of the resistance
of the nuclear matrix (the skull of the cytoskeleton) to non-ionic
detergents. This means the resulting nucei will have almost everything
EXCEPT their membranes.
A couple of years ago, I tried getting nuclei for studies of calcium
regulation. There were a couple of papers about electrophysiological
studies of nuclei which claimed to have isolated nuclei with intact membranes.
I used their methods but was never really satisfied with the results.
I will try to dig up the ref for you.
It seems that one measure you might use for assaying intactness of the
membrane is the LIVE/DEAD dyes used for whole cells. I don't know much about
the permeability of nuclear pores for dyes, but it may be that an intact
nucleus would trap cleaved calcein/AM like whole cells do.
Good luck,
Steven W. Barger, Ph.D.
Sanders-Brown Center on Aging
