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CD14 positive problems concerning monocyte derived DCs.

DC CD14 Dendritic cells Plastic adherence

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#1 kawashiro

kawashiro

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Posted 31 July 2012 - 01:39 AM

Hello,

it seems my experiments have got on a hold as a rather odd problem has arised.

I'm trying to generate dendritic cells from monocytes. This is done by isolating monocytes from PBMC from buffy coats by plastic adherence and culturing them in RPMI1640 and adding GM-CSF and IL-4. The problem that me (and others) are facing is that after the 6 days of culture and we harvest them to analyse in FACS, is that they're pretty much all CD14 positive, meaning we just have monocytes and not really DCs. We postulated that the amount of cytokines added had something to do with this development, so we ran a test with 6 different ratios of GM-CSF and IL-4, the same problem arised, all of the cultures turned CD14 positive.

This has troubled me for quite a while, and its pretty much happening all the time now (it worked very fine just a few months ago), and there have been no change in the protocol. We can exclude that there's something wrong with out cytokines (in the rare case the provider shipped faulty cytokines) as we ordered new ones to test, and we still get just CD14 positive cells. This is giving me quite a headache considering all the cytokines (aka money) invested into troubleshooting.

If anyone have an explanation to what's wrong it would be greatly appreciated.





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