Re: incubation on ice or at RT?

Dear Alireza,
 
I have just came across the staining results for the experiment that I was referring to. For different antibodies-antigens the dynamics of staining varied a bit (depending on antigen density, type of fluorochrome, etc.), but after 15 min at room temperature there was at least 90-95% of the maximum staining. The very maximum was achieved at 20-25 min, so a standard 15 min incubation was well justified. So, the staining of cell surface antigens will not be as quick as a reaction between solubilised Ag-Ab, which will also be concentration dependent.
 
With regards to early staining phases, we had 20-40% staining after 1 min, 50-75% after 5 min, and 75-90% after 10 min. I think this is what you would usually expect. We were using manufacturer's recommended antibody doses and recommended cell concentrations and volumes. The antibodies were from Coulter, who do not give much excess in a dose, so there seemed to be no overdosing of antibodies, about possibility of which Dr. Mario Roederer warned and explained in his previous message.
 
Just to add a point for the topic of choosing between staining at room temperature or at 4C / on ice, we are now considering how to translate a specific sample staining procedure from research to clinical staff. The whole staining with several antibodies including unlabelled consists of three incubation steps on ice for 35-45 min each plus several washes takes three hours. So, it will be difficult to convince clinical scientists, who are used to 15 min - no wash procedures, and also their supervisors, that it is necessary to spend three hours on staining one tube.
 
Regards.
 
Vladislav Rozenkov, M.D., Ph.D.

-----Original Message-----
From: alireza ardjmand <arardjmand@yahoo.com>
To: cyto-inbox
Sent: Tue, 30 May 2006 01:09:17 -0700 (PDT)
Subject: Fwd: Re: incubation on ice or at RT?


Dear Stuart and rozenkov
immunologists beleive that Ag-Ab reaction is one the most specific and hence rapid reaction in biology and the temprature has both good and bad effect on this phenomenun in such a way that at the beginig of the reaction higher tempratures are beneficial to the reaction whilst longer incubation helps the dessociation of epitope from paratope. Since in the first 5 min of incubation up to 90% of the reaction is almost compelet, if I were you I'd try to set the staining procedure up by incubating on ice followed by a pre incubation at RT for 5 min.
 
Yours,
Alireza
Hematology, Oncology& BMT research Cent.
Shariati Hospital,
Tehran -Iran
 
 


rozenkov@netscape.net wrote:
Date: Sun, 28 May 2006 21:44:36 -0400
From: rozenkov@netscape.net
Subject: Re: incubation on ice or at RT?
To: cyto-inbox


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' no t 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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