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There are two reasons for the non-expressing cells to be more
fluorescent than untransfected (non-DNA treated) cells. First, the
presence of positive cells, if they have a lot of b-gal activity, can
effect this: during the initial loading step (hypotonic shock),
brightly-positive cells can generate enough free fluorescein (product)
that leaks back out and into negative cells. If this is what is
happening and it is important to reduce this process, then you can try
to either (1) reduce the loading step time, say, to 45 sec; or (2)
include a low concentration of PETG (10 uM) to slow down the enzyme
enough that little product is generated during the first minute but
during the on-ice incubation (when the cells are sealed up again)
sufficient fluorescence is generated to separate the population. The
second reason is one of treatment: many transfection conditions make
cells "unhappy", whether or not DNA is actually introduced. We have
consistently found that "unhappy" cells (including confluent cells,
mistreated cells, etc.) have higher autofluorescence as well as a
higher lysosomal activity that can contribute to background
fluorescence. To control for this, your negative control should be
treated identically to the positive except for lacZ-containing DNA--it
should go through the transfection process, etc. You do not need to
have the same vector (missing lacZ) as a control, unless there is
something else on the vector that might make the cells "unhappy". (For
instance, a drug selectable marker that you are using for selection).
The use of any DNA may be an adequate control.
I am sure the Flow Community will be pleased to finally see me talk about
something that I am eminently qualified to address! :)
mr
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