Isolation of Macrophages

[Jay Mone']

Dave:

To isolate resident peritoneal macrophages, kill the mouse, and 
immediately inject the peritoneum with about 5 mls of pre-warmed HBSS 
or other media.  Keeping the needle in the peritoneum, gently massage 
the mouse to suspend the cells.  Remove as much of the media as you 
can, and place it into a centrifuge tube.  Spin the cells gently to 
pellet them, and resuspend them in about 1 ml of 0.83% ammonium 
chloride, pH 7.  This lyses rbcs.  Incubate at room temp for 2-3 
minutes, dilute with HBSS or media, and wash the cells twice.  
Resuspend the cells in MEM with 10% FBS at a concentration of 
10(5)/ml, and plate. After 24 hrs, gently wash off the non-adherent 
cells.  This procedure usually gets you >95% pure macrophages by 
non-specific esterase staining.
If you want inflammatory macrophages, simply inject 2 mls of 
thioglycollate broth into the peritoneum 3-4 days before harvest.

Good luck.  Let me know if you need more info.

Jay Mone'
Millersville University