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Excuse me if you hear a lot from me in the next few weeks. My change
in employment status has put me in the position where I'm free to post, post,
and post!
Kristi Harkins (kharkins@iastate.edu) writes:
> In reference to the lacZ question, my experience has been that the FDG or
> lacZ kits are to be used with cells in the "viable" state, and that these
> substrates are sensitive to the slightest perturbation of the cells which in
> turn causes them to leak out. Does anyone know if the cleaved product readily
> remains inside the cell once cleaved, should you then wish to "fix" the cells
> for DNA/cell cycle staining?
>
> As Jeff stated, we are interested in using
> beta-gal as a marker to distinguish one cell type from another within a mixed
> culture, so that we can then in turn identify the individual cell cycle
> distributions of each cell lineage.
Well, I went back and looked at the abstract I posted for the FACS-Gal
assay. They state that the product of FDG hydrolysis is fluorescein. It's
not carboxyfluorescein, it's just fluorescein. In my own experience and the
experience of people I know, the retention of fluorescein-like dyes is related
to the charge of the dye. Fluorescein has a single carboxyl group, and there-
fore has a very low charge. It does leak out rather fast -- and I don't think
that a loss of membrane integrity is even required for the leakage.
I assume you want to fix the cells because your cell-cycle analysis
requires it. If you do this, I'm almost certain that FDG will leak out. On
the other hand, there's a good chance that the beta-galactosidase enzyme would
also fly the coop, right? I had some luck doing cytokeratin staining with
fixed cells, but the cytokeratins are components of the cytoskeleton and are
less likely to leak when the cell membrane is ruptured.
In my experience, compounds like fluo-3 and SNARF, which are carboxy-
lated fluorescein derivatives, last quite a bit longer than fluorescein inside
intact cells. The fully-hydrolyzed forms of these compounds have two ar three
carboxy groups, which increases the net charge of the molecule. Of course,
these other compounds aren't substrates for beta-galactosidase. But it sug-
gests that, if you could make a compound that had a larger net charge after
entering the cell, it would be a better reagent for FACS-Gal. (How about an
FDG derivative with an extra acetylated carboxyl group? This would require
the activity of two enzymes on the substrate -- the beta-galactosidase and an
esterase -- but the hydrolyzed coumpound would have two carboxyl groups, not
just one.)
Now for another complete shift in gears. Yesterday I attended a
seminar given by Dr. Joseph Lipsick here at Stanford. He used a very inter-
esting method for identifying transfected cells that expressed a cytoplasmic
product. Basically, the DNA fragment he used posessed two genes on a single
RNA transcript. The upstream gene expressed the protein of interest: in this
case, an oncoprotein called myb. The downstream gene expressed CD4, which was
not normally present on the cell line he was transfecting. The assumption was
that, if the cell expressed CD4, it probably also expressed myb. The results
looked pretty good. The nice thing about this is that you can use a *surface*
protein marker to assay for the intracellular gene product. There are several
protocols which provide simultaneous surface staining and cell cycle analysis.
I used one several years ago my Dr. Michael Kasten's group at UCLA. There's
also the Rabinovitch group's method.
Good luck!
- John Ladasky
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