>One of our grad students is working with transgenic mice that have a >construct for beta-galactosidase. She has been detecting the beta-gal >with an FDG substrate introduced into the cells ( harvested from >newborn mouse thymus and spleen) with hypotonic shock. This method, >however, results in cell loss and cell damage and gives high levels of >non-specific fluorescence on the flow. > >We would like to try some other detection method - does anybody know >of an antibody to beta-gal that works in flow cytometry? Before you try this, you might try changing your FACS-Gal conditions some. We performed FACS-Gal assays on murine cells (including thymocytes, lymphocytes, and other tissues) routinely without getting either high background or loss in cell number of viability. My guess is that you don't have "clean" FDG (if the FDG solution is yellow or even mildly yellow then it has a lot of fluorescein contamination that will lead to high background; you can clean it up by bleaching it in a high-power laser (USE LASER GOGGLES)). Also, if you have a lot of cell death, you might have the concentrations off (i.e., too much DMSO in the solution). Are you adding 10 volumes of medium right after the 1 minute hypotonic load? This is very important to restore tonicity. Don't use PBS (although we have successfully used PBS for this); cells are much less "happy" in PBS than in medium like RPMI or DMEM (with serum). We have also used this same technique for measuring other enzymes (beta-glucuronidase and beta-glucosidase); we rarely saw any effect on viability (and then it was only on very sensitive cell lines; we never saw any effect on viability in primary cells). Having performed this technique on dozens of cell types for many years, I am relatively certain that the low viability you see is a problem with the application of the technique. Ask your student to try it a few more times with varying conditions! Finally, if you convince yourself that your student is using all of the right conditions (concentrations, tonicity, etc.), and you are still getting cell death, you can make the conditions more mild by increasing the tonicity during the hypotonic shock (from 50% to 60, 70%...), reducing the time of the hyptonic shock (from 1 min to 45 sec; 30 sec is the minimum "lag" before substrate loads). See the paper in Cytometry by Fiering et al "Improved FACS-Gal: ..." (Cytometry 12:291, 1991) or in Methods: A Companion to Methods in Enzymology (Roederer et al, "FACS-Gal: Flow cytometric..." (Vol 2,, 248-260, 1991). These papers provide lots of hints on tuning the assay and information on why you might get background and how to cure it! mr
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