I've been trying to cultivate BMDM into macrophages since a weak, and I'm using DMEM with 10% FBS and 5% pen strep. I changed the media three or four times and rinsed it with 1X PBS once. Although it is the eighth day of this culture, I have yet to notice a difference in the confluence. I'm using m-CSF to differentiate macrophages.
please suggest something that works for me!
I would suggest what worked great in my hands:
I hope that you are taking tibia and femur cells, followed by ACK lysis of RBCs and then washings. Culture the cells in RPMI+10% FBS instead of DMEM. I plated 2X10^6 cells/well in a 6 well plate with 60ng/ml of r-MCSF in total of 4ml media/well. I changed half the media with fresh media+r-MCSF at day 3. On day 6, I harvested the cells that showed 98% positivity of CD11b and F4/80 (markers of macrophages).
Good luck!
BMDMs are differentiated cells and are not supposed to proliferate in vitro (and in vivo proliferation of macrophages is a contested topic, probably happens only in a number of limited conditions ).
The proliferation in a typical BMDM culture happen in the first few days of the culture, when precursors (and not differentiated cells) expand and differentiate. Once differentiation occurs (to macrophages), proliferation will stop. After the 5th-6th day, the only change you can observe is a gradual loss of cells due to cell death.
Bone marrow derived monocytes (progenitors) are what you isolate from the marrow, they won't really proliferate any further, all you can do is force them to a macrophage lineage using MCSF then either pro or anti-inflammatory stimuli. Its best to make sure you count your cells and keep in mind how many you will need for your expriment. If anything after a few days you will see slight loss in cell number however after about 7 days you should notice that the cells become almost "stick or rod" like and will begin to cluster and form colonies, these are macrophages and from there you can force them to either pro or anti-inflammatory depending on the stimulus you give them. For pro-inflammatory LPS/TNFa cocktail for 24 hours works great, for anti-inflammatory an IL-4/IL-10 cocktail for 24 hours.