monocyte-derived macrophages problems - troubles differentiating macrophages from primary monocytes (Jun/28/2008 )

I'm having difficulties differentiating human monocyte-derived macrophages. This seems like a straight forward procedure but I can't seem to get it to work. I isolate PBMCs from whole blood and plate them. An hour later I removed nonadherent cells and replace the media (10% autologous RPMI) every three days. I get a good number of monocytes attached initially but after a week's time, almost none are attached (I can see a few scattered mnacrophages). Anyone have any good suggestions? Thanks!!

use a different batch of FBS or use human serum.

HI. I have also been trying to grow monocytes from PBMC isolated from whole blood. However, i don't remove the other cells and just let them culture together. But i've noticed that i don't have much adherent cells in my culture. Can you tell me how do i get more monocytes in my culture? Do you use ficoll-paque to isolate the PBMC? Do you add any mitogen in the culture to help the cells to grow? From my experience, after 7 days, if without mitogen, the PBMC starts to die off. Maybe thats why your cells detaches after one week?