Effect of fixation on GFP for FACS?

Philippe-Alexandre Gilbert aaa001 at AGORA.ULAVAL.CA
Thu Sep 23 09:31:47 EST 1999


We noticed that GFP does not leak if the cells (293) are kept in a 2%
(para)formaldehyde solution after the fixation procedure. However, we only
monitored the leakage (% positive cells and relative fluorescence mean
intensity) for a period of 96 heures after fixation. Leakage was observed if
fixed cells were resuspended in fresh PBS.

 You will have to try by yourself to verify if its working with your own
procedure.

Regards,

PAG

>>>>>>>>>>>>>>>>>>>>>><<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
Philippe-Alexandre Gilbert
Institut de recherche en biotechnologie
Conseil national de recherches du Canada
6100 avenue Royalmount, Montréal QC H4P 2R2

tél: (514) 496-6252
fax: (514) 496-6785
Internet: aaa001 at agora.ulaval.ca
----- Original Message -----
From: Vincent Metzler <vincent.metzler at zoi.unibe.ch>
To: <fluorpro at net.bio.net>
Sent: Thursday, September 23, 1999 9:56 AM
Subject: Effect of fixation on GFP for FACS?


> 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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