macrophage isolation from BM - (Sep/15/2009 )

Hi, I need to isolate some macrophages from mouse. I've been working extensively with BMDCs and am just starting with these macrophages. I found a protocol in Curr. Prot. Immunology, but there seems to be a lot of variation in the literatue so I was hoping someone who knows this area better than me could confirm this method:

Isolate femurs from mice.
Open bones and flush
Lyse RBCs
Resuspend in serum-free EMEM
Overlay suspension on lymphocyte separation medium (where to get this??)
Centrifuge 20 min 500xg, no brake
Collect cells at interface, wash
Resuspend at 5 x 10^6 c/mL in EMEM+FBS and culture 10mL in T25 flask
Incubate overnight then transfer non-adherants to larger flask containing EMEM+FBS+CSF
Feed after 4 days with new media
After 3 days, discard culture medium and detach adherant cells with dispase (can typsin work instead?)
Resuspend in EMEM-FBS and count

Thats it. How many macrophages can I expect to get with this method? Is this the best approach to preparing macrophages in vitro?

Thanks

that sounds more complicated than what we do!

You can skip the overlay on lymphocyte separation medium part. I don't know of anyone who does this. We use DMEM (high glucose) or RPMI for culturing (not EMEM). You will want to use an endotoxin-defined FBS (low endotoxin). We cultured BMM in 15% FBS.

We use M-CSF or L cell-conditioned media to grow out the BMM. The feeding schedule looks right.

Instead of using dispase or trypsin, we grow the cells on petri dishes. We remove the media and then add ice-cold PBS to the plate and use a cell scaper to remove the cells.

I am not sure of the precise numbers you will get. We typically use all the leg and arm bones so we start with a lot more cells. You should get many millions of cells with two femurs.

Macs are much easier to grow out than BMDC!