Re: Cyanine Dyes
Jacek Witkowski (jawit@amed01.amg.gda.pl)
Fri, 10 Feb 1995 16:49:02 +0100 (MET)
Adding to Howard Shapiro's comment:
I agree that using mitochondrial inhibitors can tell us if there is any
mitochondrial component in cyanine signal we use for the determination of
transmembrane potential. Howard mentions also the ability of some
mammalian cells to pump out cyanines with the help of a membrane
glycoprotein pump. The pump in question is P-glycoprotein, found on
multidrug - resistant tumor cells mostly, but also on certain
non-transformed cells, including T lymphocytes both in humans and mice
(cf my papers in JI 1993, 150:1296 and 1994, 153:658, and S. Gupta's
paper in J. Clin. Invest. 1992, 12: 451). In T cells cyanine staining
produces either a clearly distinguishable dull population, or at least a
broad shoulder of lower fluorescence, depending on the donor's age. And
the percentage of P-gp+ cells vary from around 20% in young CD4+ to more
than 90% in old CD8+ (that is in mice; in human PBL the highest values of
Pgp+ cells I saw were around 70-80% of CD8+ T cells).
On the other hand, oxonol dyes (at least DiBAC4(3) and DiBAC4(5)) are not
pumped by the P-glycoprotein (no change in fluorescence in cells with Pgp
blocked), so they remain the best choice if in doubt about possible
active elimination of the fluorochrome.
Jacek
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