Re: CD107

Hi José,

If you are using freshly thawed pbmcs for testing, I would suggest ³resting²
the cells in culture overnight prior to testing to help decrease background
for CD107.  This may also result in a decrease in background for any
cytokine markers you may be looking at.  The addition of DNase to the cell
media may lower the CD107 background by removing debris remaining from dead
and dying cells.  It is also important to ensure proper titration of the
CD107 antibody, using your same test protocol for the titration set up.

However, probably the most important step you can take to minimize the
background, is to include a dead cell discriminator in your experiment.
There are several you can use, such as EMA or PI, but we have found that the
amine dyes (Molecular Probes/Invitrogen) work best for us.  Several dyes are
available, allowing flexibility in staining panels.  Using the violet amine
dye, we were able to significantly decrease the CD107a background in our
CD4+ T cell population.

Laurie Lamoreaux, MT(ASCP), SM
Immunology Laboratory
Vaccine Research Center, NIAID/NIH


Disclaimer:
The information in this e-mail and any of its attachments is confidential
and may contain sensitive information.  It should not be used by anyone who
is not the original intended recipient.  If you have received this e-mail in
error please inform the sender and delete it from your mailbox or any other
storage devices.  The National Institute of Allergy and Infectious Diseases
(NIAID)  shall not accept liability for any statement made that are the
sender¹s own and not expressly made on behalf of the NIAID by one of its
representatives.




On 12/12/06 4:06 AM, "Jose Benito" <jbenito1@hotmail.com> wrote:

> Hi eveyone, I am working with CD107 antibody as a marker of CTL degranulation,
> in combination with several other markers. I am having problems with
> unstimulated controls, usually finding high values of CD8 cells positive for
> CD107. Is there any way to solve this problem? Even if I simply substract the
> unstimulated control from the stimulated one, I find unusually high values for
> CD8+CD107+ peptide-specific cells. So I suspect this is an artifact rather
> than real values.
>  
> Many thanks
>  
> José M Benito
> Infectious Diseases
> Hospital Carlos III
> Madrid
> Spain 
> 
> 
> FREE pop-up blocking with the new MSN Toolbar MSN Toolbar
> <http://g.msn.com/8HMAEN/2752??PS=47575>  Get it now!