Dendritic cell infection - (Sep/28/2009 )

Can anyone help me please...it'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!!!

I need the cells alive for an activation assay. thanks.

dandy.lions on Sep 29 2009, 11:30 AM said:

Hm, I know that high numbers of Mycobacteria can kill DCs. Maybe try lower MOIs?
Also: did you fix your cells before facsing? (we need to if there's mycobacteria in there). In this case you need to use fixable live/dead dyes like the ones from invitrogen, otherwise all your cells will show up dead.
I don't think we ever saw 100% infection of DCs with GFP-BCG, but a lot of our GFP postitive DCs were alive. Do you need all of your DCs to be infected? Then you might have to infect them and then sort the GFP+ ones.

Other consideration: if you don't want any mycobacteria sticking to your DCs from the outside for your assay, you will have to wash really, really throuroughly and I think our technician also uses percoll gradients to get rid of the sticky guys.

Hope that helps a bit.

Thanks so much for replying. I kind of thot that an MOI of 1 was low enough. Although 100% infection is ideal, I would be quite happy with a lower percentage so long as they live long enough to present antigenic peptides to my T cells. Have you got a protocol that I can have a look at by any chance? I just need to know that I've to the incubation times and conditions right.

Thanks again.