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there must be a problem with your methodology! We use 3T3 all
the time (i.e., weekly for the past 7 years)
with FACS-Gal, and have no problems at all. Highly-expressing
cells are usually 3-4 orders of magnitude brighter than
autofluorescence. Without hypotonic shock, there is little difference from
autofluorescence. We have no problems with chloroquine on viability,
either.
While the lysosomal enzyme activity does generate some fluorescence, it
is considerably less than that generated by the lacZ. (Assuming you
are not culturing the cells in the presence of substrate at 37C-which
simply allows lysosomal uptake of the FDG. Hypotonic shock tends to
NOT to load the substrate into lysosomes: most of it goes into the
cytoplasm).
The effect of confluence is two-fold: a general increase in lysosomal
("background") activity, as well as an increase in the presence of
"rare-brights". These are cells which appear to have large amounts of
lacZ activity (considerably more than "background"), but upon cloning
and growing up regenerate the parental distribution rather than the
"rare-bright" distribution. In confluent cultures, "rare-brights" can
be as frequent as 1-2%. I will send you references if you wish.
We have done years of experiments in 3T3 and other fibroblastic cell
lines comparing promoter strengths, stimulation indices, etc., using
FACS-gal to quantitate lacZ in these cells. It is an extremely
sensitive assay (we can detect <50 molecules of enzyme in these cells),
and with judicious use of inhibitors like PETG, has a dynamic range of
at least 5 orders of magnitude.
Are you trying to do the assay at 37C? That would be a problem, since
the fluorescein "leaks" out (pumped out) with a half-time of 3
minutes. However, at 0C it is completely retained.
Mario
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