Isolation of Macrophages
jmone at MARAUDER.MILLERSV.EDU
Tue Jun 2 06:47:20 EST 1998
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.
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