 |
 |
 |
 |
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
|
|
|
|