I am trying to get iDCs from PBMCs. After 6-7 days of incubation with GMCF and IL-4 I can clearly see dendritic cells in my flask stuck to the bottom with characteristic dendrites. After another 2 days I collect the floating cells but they appear more round shaped without dendrites. Is this ok? Usually I change the medium and factors after 6-7 days and again keep them for 2-3 days before collecting the non-adherent cells.
iDC are round-shaped and do not stick to the flask. You can check their differentiation status by checking that they are CD14low, CD1apos, HLA-DRpos, CD83neg, ICAM-1low CD86pos and DC-SIGNpos. If you activate these iDCs with LPS and IFNgamma, you will get mature dendritic cells that are CD14neg, HLA-DRpos, CD83pos and ICAM-1high. mDCs attach strongly to the flask and have a lot of dendrites.
Thanks for the explanation and suggestion. This means that whatever I am getting in floating condition should be iDC.
Michael is right. But for thought iDC show the same characteristic in FACS, they looks different in dependence of the medium for e.g. RPMI1640 or X-VIVO. Morphologic criterionns are only validity if you use the same boundering condition.
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