Simultaneous FDG and surface markers

Mario Roederer Roederer at Darwin.Stanford.EDU
Tue Mar 26 18:49:22 EST 1996


>   Has anyone had substantial experience staining the same
>   population of  cells for B-galactosidase activity using
>   FDG AND cell surface proteins  using direct or indirect
>   detection?  

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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