Effect of fixation on GFP for FACS?

Alistair Forrest zzalforr at fox.uq.net.au
Thu Sep 23 21:02:32 EST 1999


G'day Vincent,
	We routinely use 70% ice cold ethanol for fixation. However
straight GFP will leach out. If you are using a GFP fusion then generally
it will not leach out, although it depends how big the fusion is. Our
fusion is approx 50 kDa.
	We also found some of our fusions did very nasty things to our
cells. This may be because we were using the original EGFP instead of a
destabilised version, the increased stability and expression levels of our
full length construct seemed to cause apoptosis.

cheers, 

	Al Forrest
	


Vincent Metzler <vincent.metzler at zoi.unibe.ch> writes:

>Hi everybody,

>I'm new in the field of FACS analysis. The problem I want to deal with
>is the following:

>I need to determine transient transfection efficiencies by sorting the
>cells (FACS on HeLa cells transfected with a GFP-expressing plasmid).
>But now I'm wondering if the HeLa cells will loose the GFP-fluorescence
>after fixation (paraformaldehyde, I suppose) and storage (how?) until I
>collected enough samples to do the FACS. I have to fix these cells also
>because of biosafety considerations: they will be transfected with an
>HIV-based vector.

>Thanks for your help. Vincent

>vincent.metzler at zoi.unibe.ch
>University of Berne, Switzerland




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