PI and GFP

csg admin csg.admin at amgen.com
Tue Feb 25 21:46:38 EST 1997


Mail*Link® SMTP               RE>PI and GFP


Wendy,
We have found that ethanol fixation kills the gfp.  We have used some of
the non-alcohal fixes such as streck, orthopermeafix but the CV's are not
very nice with pi.  You might want to try Hoechst 33342 which does not
require fixation and permeablization for the DNA/surface analysis. 

Good luck
Rochelle Diamond
Caltech Flow Cytometry/Cell Sorting Facility
diamond at cco.caltech.edu


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 at bcm.tmc.edu
>


------------------ RFC822 Header Follows ------------------
Received: by amgen.com with ADMIN;25 Feb 1997 13:35:19 -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 NAA02014;
	Tue, 25 Feb 1997 13:21:23 -0800 (PST)
Received: by castle-smtp.amgen.com; id NAA24825; Tue, 25 Feb 1997 13:21:22 -0800
Received: from flowcyt.cyto.purdue.edu(128.210.60.31) by castle-smtp.amgen.com via smap (V3.1)
	id xma024822; Tue, 25 Feb 97 13:21:03 -0800
Received: by flowcyt.cyto.purdue.edu (940816.SGI.8.6.9/930416.SGI.AUTO)
	for cyto-sendout id PAA27980; Tue, 25 Feb 1997 15:22:27 -0500
Received: from accord.cco.caltech.edu by flowcyt.cyto.purdue.edu via ESMTP (940816.SGI.8.6.9/930416.SGI.AUTO)
	for <cytometry at flowcyt.cyto.purdue.edu> id LAA25901; Tue, 25 Feb 1997 11:27:02 -0500
Received: from localhost by accord.cco.caltech.edu with SMTP
	(8.6.12/DEI:4.45) id IAA12531; Tue, 25 Feb 1997 08:19:28 -0800
Date: Tue, 25 Feb 1997 08:19:27 -0800 (PST)
From: "Rochelle A. Diamond" <diamond at cco.caltech.edu>
X-Sender: diamond at accord
To: cyto-inbox
Subject: Re: PI and GFP
In-Reply-To: <Pine.SOL.3.91.970224102100.7570A-100000 at watson.bcm.tmc.edu>
Message-ID: <Pine.SUN.3.95.970225081211.11917B-100000 at accord>
MIME-Version: 1.0
Content-Type: TEXT/PLAIN; charset=US-ASCII






More information about the Cytometry mailing list