Monocyte derived Dendritic cells - (Apr/11/2008 )

hi there,
I am trying to derive dendritic cells from human monocytes by plastic adherence method (we cannot afford to do MACS). I AM TRYING IT SINCE SUCH A LONG TIME BUT NO SUCCESS. i remove the non adherent cells after two hour adherence of the PBMCs.then add GMCSF and IL4 (THIS TIME I HAVE TRIED WITH 50 NG/ML OF EACH) on alternate days.when i see my cells under microscope i can see many comma shaped and irregular cells which i think are monocytes but when i try to visualise the population at the end of 7th day using CD14 and CD11c there was not a single cell.
i have also run a control (in which i did not add cytokines). it has got many macrophages in it.
could it be that i have to use higher conc of growth factors? can somebody please tell me what is the half life of these cytokines? may i need to add them daily.
pleeeeeeeeez help

goofy17

http://www.ncbi.nlm.nih.gov/pubmed/12169207

can this help? I read this some time ago, it is a Romanian paper but still quite good

Apoptosis in the immune system: Fas-induced apoptosis in monocytes-derived human dendritic cells.Nat R, Radu E, Regalia T, Popescu LM. J Cell Mol Med. 2002 Apr-Jun;6(2):223-34