Re: A problem with the sort of GFP transfected cells on the FACS DiVa

Jochen,

> I would like to ask your input / help with a problem, we recently 
> faced twice trying to sort GFP transfected cells (once mouse bone 
> marrow and once adherent cultured human cell lines.
>
>  Running the samples we found only very few and very weekly 
> fluorescing cells. But whenever we stopped the sample flow during 
> acquisition, the last “burst” of cells, showed up in the region, where 
> we normally found strongly positive cells. This effect was always 
> identical (when we switched on and off every few seconds or stopped 
> directly after a 30 minutes run). With the bone marrow cells 
> backgating showed a scatter pattern as we expected.
>
> The same phenomenon was seen, when we used the sample boost during 
> acquisition: The first “burst” of the boosted cells seemed to be 
> strongly positive. Then the pattern went back to normal of course with 
> the low reolution of a boosted sample.
>

Your problem is a bit puzzling but I wonder if it relates to our 
experience with some GFP samples.  Often the fluorescent protein leaks 
out of the transfected cells and can stain other non-transfected cells 
in the tube.  However, it also sits around in the suspending medium and 
that alone can give rise to high fluorescence levels that are 
*apparently* associated with cells.  If you have an instrument that 
shows a detector pulse display, you can see whether the background 
level of the GFP detector trace (i.e. away from the initial triggering 
pulse) rises with your "boost" switching and correlates with the 
appearance of your "positive cells".

Frank Battye.

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