[Immunology] Re: Immunology

Cornelius Fritsch C.Fritsch at dkfz-heidelberg.de
Tue May 23 02:46:55 EST 2006


On Mon, 22 May 2006 22:58:58 +0200, Nischal Padala <npadala at gmail.com>  
wrote:

> Hi!
>
> Im trying to do a mouse T cell proliferation assay. According to the
> literature out there one should be able to induce CD4+ T cell  
> proliferation
> by using anti cd3 and anti cd28. But I cant seem to get this to  
> work...i.e.
> the cells are not multiplying (im measuring proliferation by using Brdu  
> and
> FACS). I have tried different concentrations for cd3 (from 3ug/ml to 25
> ug/ml) and cd28 (2ug/ml to 8ug/ml). I also tried them as both platebound  
> and
> soluble antibodies. I dont know what I am doing wrong...any  
> help/suggestions
> or protocols would be greatly appreciated!
>
> Thank You
> N K


One common problem is the crosslinking of the antibody. When you use  
soluble antibodies, you need to provide some crosslinking by including  
either an anti-mouse antibody or some protein A. When plating the  
antibodies to the culture dish, you need to wash extensively before adding  
the cells to remove any unbound antibody.

When crosslinking is insufficient, you get a lot of monovalent binding  
between antibodies and their target, which will provide insufficient  
signals.

Hope that helps,

Cornelius


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