Re: incubation on ice or at RT?

Hi Stuart,
You did not mention whether you stain for the same time at RT as on ice or longer. We
used to stain for 15 min at RT or 30 min at 4C in a refrigerator (depending on other
reasons for RT or 4C) and believed that that would give comparable levels of staining,
because at lower temperatures Ag-Ab reaction kinetics slows down. On ice (0C) would
require longer staining.
This will depend on concrete antibody and antigen, Ag density, Ab concentration,
affinity, etc., but generally in our experiments we have shown that 15 min at RT or 30
min at 4C brings staining close to the plateau, although some additional staining still
occurs after that.
So, if you stained for 30 min both on ice and at RT you had more staining in the latter
case.
I think the classical way of staining for research was 30-40 min cold (and we used to do
this earlier), but recent simplifications of the staining protocol introduced by antibody
companies for routine diagnostics (e.g., whole blood, no wash) included RT staining (and
we currently use it). That reduced processing time (15 min staining is enough) and made
procedure more reproducible, because everything is done at ambient temp without
temperature swinging between original sample storage, ice box, centrifuge, bench, etc.
Preventing capping and shedding were the reasons for low temp antibody staining, but
sodium azide in antibodies and all media during processing also helps with this.
Of course, for some specific scientific applications staining at 4C or on ice may be
optimal, but staining time needs to be adjusted respectively.
Regards.
Vladislav Rozenkov, MD, PhD
Melbourne
Australia
-----Original Message-----
From: Stuart Berzins <berzins@unimelb.edu.au>
To: cyto-inbox
Sent: Fri, 19 May 2006 10:46:46 +1000
Subject: incubation on ice or at RT?


We have always stained lymphocytes for FACS on ice because of the standard arguments
relating to reduced on-off rates, anti-capping, preventing antigen down-regulation etc
etc. However, we recently noticed that the intensity of signals for chemokine receptors
was much higher (sometimes a log) when we stained at RT compared to on ice. Sometimes a
far greater proportion of the cells tested positive when stained at the higher temp. We
then tested other antibodies and also a CD1d-tetramer reagent (to detect NKT cells) and
surprisingly found that most of these (including anti-CD3, anti-CD8 and the tetramer)
also gave much brighter signals when stained at RT, although the proportion of positive
cells remained largely the same. Some mAbs had signals of identical strength, but none
were worse at RT. We are struggling to find a reason not to stain at RT. Cell viability
is an obvious concern, but perhaps we could stain at RT but then wash and store the cells
on ice so that they are only at the higher temp for 30min at most. Can anyone explain why
we are getting better signals at RT for virtually all our reagents and provide some
advice as to whether we are 'safe' not to stain on ice. 
As a sideline, I remember some correspondence on this topic that said staining on ice was
not as good as at 4 degrees. I can't find the original literature, but could this be part
of our problem? 
Stuart 
-- Stuart Berzins Ph.D, 
Research Fellow, Godfrey Laboratory, 
Department of Microbiology and Immunology, 
The University of Melbourne, 
Parkville 3010, 
AUSTRALIA. 
email: berzins@unimelb.edu.au 
Ph: +61-3-8344-5704 
Fax: +61-3-9347-1540 
Mobile: 0427 849 123 
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