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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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why dendritic cells died in phagocytosis assay?
Posted by: lovepotatoes (IP Hidden, New member, 1)
Date: January 9, 2008 06:39PM
I am using GFP-E.coli to detect dendritic cell phagocytosis, but I have a big problem.
Here is my protocol: 1 million bone marrow derived dendritic cells in GM-CSF-supplemented DMEM; 50 million live GFP-E.coli in PBS; Coculture them together in 1 ml GM-CSF-supplemented DMEM at 37 degree centigrade for 1 hour; Wash them with PBS once; Resuspended in 500 ml PI-containing PBS and analyze by FACS to detect the GFP-positive cells. But >90% cells were PI-positive, meaning they were all dead. What is the problem? Why did these dendritic cells die? PS: I can not inactivate the E.coli by heat or fix them with ethanol, PFA, etc because the GFP will also be inactivateed after these treatments. I appreciate any of your help!
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