Mail*LinkŪ SMTP RE>PI and GFP
Hi Wendy,
I posted a similar query about a month ago and have had some success
thanks to the suggestions I received.
We were having similar problems in that in order to get a decent PI
profile we wanted to use ethanol fixation but ethanol alone caused a great
reduction in the GFP positivity (both percentage and fluorescence
intensity). Like you, we found that CD2 or CD8 co-transfection was fine,
only GFP suffered.
We received several suggestions including changing the GFP to one that
had, for example, a Golgi membrane binding to minimise loss from the cells
after fixation. However, most suggestions concerned the fixation
procedure.
The approach we are now using is to lightly fix in paraformaldehye to fix
the protein in place, but not overfix so as to make accessiblility to the
DNA difficult. Then a postfix in ethanol followed by propidium iodide
staining. There is still a reduction in fluorescence intensity which we
may have to live with but we can now get an acceptable DNA histogram
showing GFP positive fluoresence in FL1.
OK, a few more specifics about the staining:
We were using human osteosarcoma cells transfected with GFPA.
48hrs after transfection, cells were fixed in 0.25% paraformaldehyde for 5
minutes.
Wash in PBS.
Fix in cold 70% Ethanol for 30 minutes
Wash in PBS x3
RNase (100ug/ml, 10 mins RT), then 50ug/ml PI
G1 CVs were around 5.0.
Hope this is of some help!
Any queries can be directed either to me
or to Clare Hughes (Clare.Hughes@icrf.icnet.uk)
Derek
****************************************************************************
* Derek Davies Voice: (44) 0171 269 3394 *
* FACS Laboratory, FAX: (44) 0171 269 3100 *
* Imperial Cancer Research Fund, e_mail: davies@icrf.icnet.uk *
* London, UK *
* *
* Web Page: http://www.icnet.uk/axp/facs/davies/index.html *
****************************************************************************
On Mon, 24 Feb 1997, Wendy D. Schober wrote:
>
> We are attempting to optimize the procedure to label transfection along
> with cell cycle in HeLa cells. So far we have co-transfected with CD20
> and then labeled the surface with CD20-FITC. We found 5-13% CD20
> positive with good PI patterns after the usual 70% ethanol fixation.
> We used the same cells and fixation procedure with GFP as the reporter and
> hoped to find similar results. Unfortunately, there were few cells GFP
> positive and what may be there are of very low intensity, barely over
> background. We are looking for suggestions for improving our GFP and keeping
> good PI patterns. The GFP is from Invitrogen and they have been somewhat
> helpful, but have not done this with PI.
> Is fixation causing a problem?? If so, is there a better procedure which
> will permeabilize and fix to get both GFP and PI to work?? Ultimately, we
> want this to work in some fibroblast lines, but HeLa is our control.
>
> Thanks in advance for any hints.
> Wendy Schober
> wschober@bcm.tmc.edu
>
------------------ RFC822 Header Follows ------------------
Received: by amgen.com with ADMIN;25 Feb 1997 11:40:58 -0800
Received: from castle-smtp.amgen.com (castle-smtp [138.133.19.51])
by www-int.amgen.com (8.8.5/8.8.5) with SMTP id LAA23844;
Tue, 25 Feb 1997 11:27:01 -0800 (PST)
Received: by castle-smtp.amgen.com; id LAA21289; Tue, 25 Feb 1997 11:27:01 -0800
Received: from flowcyt.cyto.purdue.edu(128.210.60.31) by castle-smtp.amgen.com via smap (V3.1)
id xma021282; Tue, 25 Feb 97 11:26:40 -0800
Received: by flowcyt.cyto.purdue.edu (940816.SGI.8.6.9/930416.SGI.AUTO)
for cyto-sendout id NAA26906; Tue, 25 Feb 1997 13:30:01 -0500
Received: from europa.lif.icnet.uk by flowcyt.cyto.purdue.edu via SMTP (940816.SGI.8.6.9/930416.SGI.AUTO)
for <cytometry@flowcyt.cyto.purdue.edu> id KAA25351; Tue, 25 Feb 1997 10:02:37 -0500
Received: from localhost
by europa.lif.icnet.uk with SMTP(5.65v3.0/6.2); Tue, 25 Feb 1997 14:55:08 GMT
Sender: davies@icrf.icnet.uk
Date: Tue, 25 Feb 1997 14:55:08 +0000 (GMT)
From: Derek Davies <davies@icrf.icnet.uk>
X-Sender: davies@europa.lif.icnet.uk
Reply-To: Derek Davies <davies@icrf.icnet.uk>
To: cyto-inbox
Subject: Re: PI and GFP
In-Reply-To: <Pine.SOL.3.91.970224102100.7570A-100000@watson.bcm.tmc.edu>
Message-Id: <Pine.OSF.3.93.970225095941.4598A-100000@europa.lif.icnet.uk>
Mime-Version: 1.0
Content-Type: TEXT/PLAIN; charset=US-ASCII
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:49:27 EST