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Yes, we have done this extensively!
> That hypotonic FDG loading sometimes has a tendency to
> render the cells quite fragile. My inclination in the
> past was to refrain from excessive centrifugation and
> resuspension post-FDG loading. Therefore, my instincts
> tell me that surface staining after hypotonic loading
> should probably not be considered.
We don't find a fragility problem in all the cell types we have used. In fact,
many cell types can withstand up to 2-3 min of hyptonic loading (increasing the
FDG loaded and therefore the resulting sensitivity). However, if you do have a
problem, you can use a less hypotonic medium. i.e., instead of a 50% tonic
shock, you can try 60%, or 70%. The amount of FDG loading (and therefore the
sensitivity of the assay) is proportional to the difference in tonicity from
100% (I can give you reference if you are interested). You can also do a
shorter load time. The amount of FDG loaded is proportional to the time in
hyptonic medium minus 30 seconds; i.e., there is a 30 sec lag before FDG enters
the cells.
> Am I on the right track? Does "surface stain first,
> FDG-load second" sound like a reasonable strategy?
If it works, use it. However, we always do the other way around with excellent
results.
mr
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
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