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Subject: Time: 10:14 AM
OFFICE MEMO FDG Staining Date: 13/9/96
Has anyone out had any experience with flow cytometric analysis of b-gal
activity using FDG to detect transient expression of b-gal constructs? We are
following the protocol published in Cytometry 12:291-301 (1991) by Fiering et
al (and have also tried the slightly different protocol of Berger et al
Cytometry 17:216-223 (1994)). While the method certainly works we are having
trouble quantitating the results - assay of positive control cells results in a
very nice positive peak 1-2 logs higher than the negative control, but mixing
the two cell types before assaying results in a peak in the middle rather than
one neg and one pos peak. The problem appears to be leakage of the fluorescein
from positive cells into the medium during the hypotonic shock step (the
medium becomes bright yellow during the 60 second incubation at 37oC). We
have tried altering various parameters such as reducing the number of cells (we
usually use 10^6 cells per sample), reducing the length of the hypotonic
shock, and reducing the concentration of FDG loaded into the cells. However,
none of these have had any effect. The construct we have been using as a
positive control is pCMVbgal - it may be that the level of bgal after
electroporation into the cells (a suspension cell line) is so high with this
promoter that we will never get quantitation. We are trying weaker promoters
to see if this is the problem. The FDG solution I am using is a bit old (made
up two years ago, stored at -20 with multiple freeze-thaws) so maybe that is
another variable. I have ordered in a new stock just in case. An alternative
is to try the C12-FDG as there is evidence that it is a better substrate for
mammalian cells. I would really like to get this method quantitative, and
prefer it to the usual colorimetric b-gal assays at is more sensitive and I can
gate out the dead cells by PI staining (electroporation of the cell line I am
using results in a very low viability). Any thoughts/comments would be
appreciated.
Thanks in advance,
Michelle Miller
Johnson and Johnson Research
GPO Box 3331
Sydney 2001 Australia
Ph 61 02 9360-9377
Fax 61 02 9360 9813
Email PRI-SYDNEY[Michelle__Miller]@immedia.ca
(note this address is case sensitive, the square brackets and double
underline in the middle of my name)
Alternative Email: jjphar13@angis.su.oz.au
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