Re: Adjusting staining - Summary and Thanks

There are no hard and fast rules for amounts of antibody needed to  
stain large numbers of cells.  You can generalize: most antibodies  
that are properly titrated for 1 million cells will not show any  
decrease at 10 million, and only mild decrease at 100 million.	The  
book chapter that Aaron Kantor and I wrote years ago (I'll send to  
anyone who requests... my annual advertisement) details this.  For a  
full explanation of these issues, and a brief description of how to  
titrate antibodies properly, see Kantor, A. and Roederer, M. (1997).  
FACS analysis of lymphocytes. In: Handbook of Experimental Immunology  
(Fifth Edition), Herzenberg, L. A., Weir, D. M., Herzenberg, L. A. and	
Blackwell, C. (ed.), Blackwell Science, Cambridge, pp 49.1-.13.

When you titrate on a million cells, do NOT use "excess".  Use the  
minimum amount of antibody that gives you the full fluorescence (if  
you want saturation).  If you're not concerned about MFI, then you can	
use less antibody -- that amount that gives you the best separation  
for your needs.  Excess antibody is wasteful and can lead to increased	
background.

You have to be careful though.	The affinity of the antibody will  
affect this.  Almost counter-intuitively, the very high affinity  
antibodies will be much more adversely affected by cell number than  
low-affinity antibodies.  This is because high affinity antibodies can	
be used at very low antibody concentration -- meaning that when you  
increase the cell number dramatically you will use up all the antibody	
(not in excess) and lose MFI.  On the other hand, saturation with low- 
affinity antibodies only occurs at high concentrations, so you will  
always be in excess.

When we stain 100-200 million cells, we often increase the amount of  
antibody 5-fold just to be safe.  If you will routinely use 100+  
million cells, then do a quick 3 or 4 point titration -- just by  
comparing to your full titration at 1 million cells you will know what	
concentration to use.

Note that the staining volume is relevant only with regard to the  
amount of antibody to use.  The only pertinent factor with antibody  
staining is the antibody concentration.  Thus, if you stain in twice  
the volume of your titration, you MUST use twice the amount of	
antibody (even if you're staining only 1 million cells).

Finally--there is NO SUCH THING as an antibody titre expressed as  
amount "per million cells".  This is completely incorrect and has no  
basis in reality.

mr

On Jun 26, 2008, at 2:59 PM, hadi aslan wrote:

> Dear all,
>
> First, many thanks for all who have invested time to reply to my  
> questions!
> I would like to summarize my conclusions from all flowers' answers:
>
> 1. Titration with small numbers of cells (1x10^6 cells) should be  
> the first
> step, to find the optimal staining for the specific cells and  
> antibody. The
> antibody should in excess and not the minimal amount that is	
> adequate for
> good staining.
>
> 2. The Abs dilution is the critical point and shouldn't be changed  
> in this
> kind of adjustment.
>
> 3. The cell number might be increased while keeping the same staining
> volume. The extent of increasing the cell number was not clear  
> though, in
> some answers I got up to 5-fold increase and in some even more (about
> 50-fold).
> I would put it in the middle and say that increasing the cell number	
> by
> 10-fold might still be reasonable.
>
>
> If anyone have any suggestions/additions, please write them.
>
>
> Hadi Aslan
>
> PhD, DMD
> Skeletal Biotech Lab/Prof. Dan Gazit
> The Hebrew University of Jerusalem- Hadassah Medical Center
> PO Box 12272, Jerusalem 91120 Israel
> Tel: 972-2-6757625
> Fax: 972-2-6757628
> Mobile: 972-545-909208
> Web site: http://gazitlab.huji.ac.il
>
>