I am generating mdDCs (human). I am using 50ng/ml and 100ng/ml IL4 and GMCSF (respectively) added at day 0, 3 and 5. Colleagues in my lab used these concentrations for their work and always ended up with no CD14 expression on mdDCs at day 7. I am maturing my mdDCs at day 6 so when I do QC I always have some CD14 positive cells! It is usually ~15% of cell population but once I had up to 35%! mdDCs were CD209, CD11c and CD1a positive (95-100%). Would CD14 drop at day 7? How sure can I be that those cells are actually mdDCs rather than macrophages?
I will appreciate your comments!
Some CD14 expression on MDDCs is totally normal (they develop from monocytes, so...). If you have high expression of MHC-II, you can be at ease.
Maybe you´re collecting some adherent cells together with the loosely adherent cells, which are probably macrophages. If that´s the case, on your FACS figure you´ll see a well defined population of CD14+ cells. Either way, the expression of low levels of CD14 on DCs is not unusual and you shouldn´t worry about it.
Additionally, if you are activating your MoDCs with LPS it's normal to see CD14 upregulation in your cells! If you have a control monocyte/macrophage you will probably find those cells CD14high and the MoDCs CD14low!
In vitro generated MoDC generally maintain the CD14 marker low.
The CD209 (DC-Sign) is a good marker, as the CD1a, to monitor the maturation of MoDC
But the most significant marker is CD208 (DCLamp) that fully replaces the CD68 which disappears completely at the end of MoDC maturation.
Did you use Fc Receptor block prior to antibody staining? Could it be non-specific binding of anti-CD14 to Fc receptors on your antigen presenting cells?