why dendritic cell died in phagocytosis assay? - (Jan/09/2008 )

Hi, guys,

I am using the GFP-E.coli to do the dendritic cell phagocytosis assay. But after coculture DCs with E.coli at 37 degree centigrade, nearly 90% DC died. I am crazy for this. Here is my protocol:


1 million bone marrow derived dendritic cells (Day 9 culture) with 50 million alive GFP-E.coli, at 37 degree centigrade for 1 hour.
After that I washed them with PBS and spin down at 500 rcf for 5 minutes. Then I use PI to exclude dead cells and detect GFP by FACS.

But >90% cells were dead, and I have repeated this for several times!

What is the problem? Anyone knows why and how?

LPS (from E.Coli) --> TNF-alpha release (from Macs)---> cell death??

LPS leads to Interferon (alpha or beta) release and cell death.

So should I wash the E.coli for several times to dilute the LPS as much as possible before adding into cell suspensions?
Anyone did his kind of experiment?

I can not use heat-inactivation or fix the E. coli because the GFP will be inactivated and the bacteria will not be green after such treatment. So any other good ideas?

LPS is very potent and is a part of the bacteria, I am not sure if you can remove all. Can you do this and titrate E.coli dosage to find a useful range?

I did this experiment with different cell/E.coli ratio: 1: 10, 1:25, 1:50 and 1:100. The cells in the 1:10 group were relatively better, but also about 60% were dead. I am not sure if the lower ratio, meaning 1:1 or 1:5 is better or not. The lower cell/E.coli ratio may pose another problem: whether FACS can detect such few GFP-E.coli in cells. Because I think the less the E.coli added into cell suspension, the less the E.coli could be uptaken by dendritic cells, and the weaker GFP detected.