I have isolated PBMC from human blood and allowed to adhere to plastic surface for 3 hours and then washed with media to get rid floating cells.Then adherent cells were stimulated with IL-4 and GM-CSF (20ng/ml) each. Then we allowed to grow for 7 days .Now we want to look for immature dendritic cells . So kindly suggest me some marker which specially found on immature Dc derived from cultured PBMC.
THANK YOU
You can look at the attached article for discrimination of iDC.
Good luck
You might need to feed your cells at day 3 - check if the media becomes very yellow.
In general check for loss of CD14 and Cd1a positivity - coupled with morphology and FSC/SSC profile on flow would be the minimum you need.
For maturation HLA-DR and CD86 usually work very well.
Then depending on your specific question there is an array of activatory/inhibitory receptors, chemokine receptors etc to look at.....
Elizabeth do you have any articles on the array or activatory/inhibitory receptors?
Hello,
iDC usually have a higher CD209 and ILT3 expression, compared to mDC. They should be CD14-, CD11c+, CD80low, CD40low, CD83-
DCsign, CD209 is the best candidate: not present on monocytes and strongly positive on GM + IL4 monocytes after 6 days of culture.
Hello ,
we check the change of CD14+ and CD1a- and make sure that are CD83-
trouble with CD80 we observe different clone cause extrem different results
Hi Suchitra,
PBMC differentiated to iDC should be CD14-, CD1a-, CD83-, CD11C+, CD209+, low CD40, CD80 and MHC Class II. The expression of CD86 and MHCII is intermediate in iDCs but becomes very high in mature DCs. You can use CD14 and CD1a as control of differentiation. If your cells show high expression of CD40, CD80, CD86 and are CD83+ they already matured.
CD11c is not a great marker for MDDCs differentiation - better used in blood as a DC marker in combination with Lineage 1 - as CD14+ cells can be CD11c+ too. CD209 is a good marker of DC differentiation too - as is upregulated (by IL-4) on culture of the monocytes. Best way to tell if the cells are activated is actually by looking at them; single same sized cells, likely immature; clumped colonies of cells - then they are mature! Check what happens to the cells 4 hours after adding LPS - clumps form if the cells are maturing - integrins are upregulated. So look at them first and then confirm this by flow - for HLA-DR, CD80 etc
CD209 is actually the best differentiation marker.
Dora is a good marker of immature DC, it is strongly decreased after CD40 activation.
CD208 is the final maturation marker after activation LPS or CD40.
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