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Dead Dendritic cells ... please help - (Sep/27/2009 )

Can anyone help me's quite urgent. I have tried infecting dendritic (derived from macrophages isolated by MACS CD14) cells with GFP- Mycobacterium. FACS stains of the 'infected' cells showed that not only was the infection partial even at MOI's ranging from 1,10 and up to 50, but all the cells that appeared positive for GFP also stained in the dead cell channel. This should be a straightfoward protocol so I think I'm doing something wrong. Has anyone got a really detailed protocol that's so basic that it tells you what plates/tubes etc you need to use and exactly how to use them. Please help me!!!


PS.. I need to use the infected DC's in an activation assay, so I need them alive.

Thanks again.