moncyte derived macrophage - (Aug/21/2009 )
Hi,
I try to get monocytes derived macrophage. Monocytes were isolated from PBMC by ficoll and CD14 Microbeads. Monocytes were cultured in DMEM with M-CSF, heat inactivated human serum,ciprofloxacin and geatamicin. After 2 days stimulation, I found there are still a lot of unattached cell(I seed cells to 24 well plate at 1.2*106/well ). I thought they should turn to adherent cells after overnight stimulation and be fully differentiated at 7 days culture. I wonder how long monocytes should completely attach to plate?
Thank you very much for any suggestion and reply
We routinely isolate bone marrow macrophages and we seed in 250 ml culture flasks. After culturing for overnight in the presence of MCSF we do transfer the medium containing unattached cells to another flask and culture overnight before adding fresh medium. Normally we seed the macrophages from one mouse on to 250ml culture flask. I think u need to increase the area for adherence.
Hope this helps...
virus on Aug 21 2009, 09:26 AM said:
I try to get monocytes derived macrophage. Monocytes were isolated from PBMC by ficoll and CD14 Microbeads. Monocytes were cultured in DMEM with M-CSF, heat inactivated human serum,ciprofloxacin and geatamicin. After 2 days stimulation, I found there are still a lot of unattached cell(I seed cells to 24 well plate at 1.2*106/well ). I thought they should turn to adherent cells after overnight stimulation and be fully differentiated at 7 days culture. I wonder how long monocytes should completely attach to plate?
Thank you very much for any suggestion and reply
Monocytes should adhere right away but become nonadherent later, depending on the culture medium and growth factors. We have some cultures of human monocytes that are still mixtures of adherent and non adherent cells after 2 weeks of culture, but that is not typical.
We recently had a customer who purchased our negatively selected monocytes and had problems. He discovered it was a bad serum lot. Can you try another lot of serum?
After reading about this topic, it seems to me that there can be great differences between experimental conditions that seem to be very similar. Maybe there are significant differences between individual mice/men, too.
According to most sources, murine monocytes should be adherent after 40 minutes - one hour. My own experience is that coating the flask with lysine helps a lot, but other people say that monocytes should adhere very strongly on bare plastic as well.
Concerning the point on differentiation, I heard the opinion that differentiation from monocyte to macrophage begins immediately with adhesion of the monocytes on the culture vessel. I don’t know if this is true. 
By the way, how do you isolate the PBMCs from the animal? Could they get damaged before you continue with the other steps?