T cell activation. - (Nov/14/2005 )

Hi!
I tried Brdu detection in activated T cells after 3 days of culture, but i failed.

I have permeabilized the cells, refix, DNAse treated, but still I am not able to see any BRDU staining in Flow cytometery

I need some help and hints to make this succesful.

Are you sure your cells are actually proliferating? If they are, the chances are it's a permeabilisation problem. What are u using as a permeabilisation agent? After surface staining, i fix with 2% PFA on ice for 20 mins, then permeabilise overnight with 0.1% Tween and 0.5% PFA, i have tried loads of ways of permeabilisation for BrdU and this works the best for me. Hope this helps.