>well it doesn't make a lot of sense to me either but it works. The GFP
>setting is low -however even if I could lower it some more that would not
>help. With these samples the pos and neg pops merge as they get scrunched
>up together- i.e the neg pop moves up and the resolution of the neg pop
>gets tighter. The ND filter used is from Cytomation. (UVND 0.6)
It does not happen in the absence of Hoechst and it does not occur with all
samples. Same problem is visible per microscope so its not the Sorter. I
tthought the problem might lie with the preparation of the Hoechst: e.g.
some people used DMSO instead of water to dilute Hoechst, others ancient
aliquots, others stained with Hoechst which had loads of crystals floating
around in it, another froze the powder and kept rethawing that to make a
fresh aliquot each time .... there must be 50 ways to ignore a protocol.
But even when they get it right the problem occurs.
So to quote Howard :
. "Hoechst and DAPI do form some yellow fluorescent products
with materials other than DNA (and I'm not sure anybody knows what they
are) in some bacteria (and, if I remember correctly, also some plant cells)."
In that case could it be related to the type of fly cells being
analysed??? There's very little info available involving FCM and
Drosophila or Mosquito.
With insects - especially if you have to dissect them first to remove the
bits you need- there is very little material and hours of work involved-
therefore being able to "correct" this with the ND filter was a relief as
on the first occassions all I could say was- too bad try again.
At present I'm trying to design a protocol for bulk preparation of fly
cells in order to have enough material to address encountered problems
which means I will have to see those flies in the flesh sooner or later.
Regards
Ann
>
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