Macrophage Isolation: When things get cloudy

Macrophage Isolation: When things get cloudy

Macrophage Isolation:
When things get a little cloudy

by Heather Buschman

Macrophages are dedicated phagocytes that love nothing more than seeking out infection or inflammation and gobbling up bacteria, dead cells, and other debris. Once activated, these innate immune defenders also release chemokines and cytokines that trigger an inflammatory cascade.

In the lab, macrophages are used to test all kinds of things, from phagocytosis to trans-endothelial migration. One of the most common ways to obtain macrophages is to elicit them from the mouse peritoneum. First, healthy mice are injected intraperitoneally (i.p.) with 3 mL of 3-4% thioglycollate. This causes inflammation, triggering monocyte differentiation and localization to the body cavity. Thus, this method results in a higher yield of macrophages than techniques that rely on collecting only resident macrophages. However, thioglycollate-derived macrophages are “activated”, and therefore are not appropriate for every experiment. (Alternatively, murine monocytes can be collected from the bone marrow and differentiated into macrophages, with high yields and little activation.)

After 3-5 days, the thioglycollate-injected mice are sacrificed, injected i.p. with 5-10 mL of sterile, ice cold phosphate buffered saline (PBS), and gently massaged. Then the skin is carefully snipped and peeled back to reveal the intact peritoneum, distended with fluid. At this point, a large gauge needle and syringe are used to draw back out as much of the injected PBS as possible, without disturbing the membrane or organs. This extraction should be cloudy with white cells, but mostly free of red blood cells. Keeping this solution cold, the cells are gently centrifuged and rinsed with fresh cold PBS several times before resuspending in tissue culture media.

As discussed in the mouse peritoneal macrophages isolation forum at Scientist Solutions®, the easiest method for separating the macrophages from the other white blood cells is by simple adherence. When plated in a tissue culture dish, the macrophages will stick overnight and the rest of the cells can be rinsed away the next day. To increase macrophage yields, several posterssuggest “aging” the thioglycollate. Nobody seems exactly sure why, but everyone agrees that autoclaved thioglycollate stored for a week to several months just works better. Some forum participants also report better results extracting with 16-30% sucrose rather than PBS.