macrophages primary culture

[Michael T. Kleinman]

We routinely obtain alveolar macrophages by bronchoalveolar lavage from
rats.

After anesthesia to a surgical plane with 75 mg/kg pentobarbital we
isolate the trachea, asceptically, and install a cannula via a
tracheotomey.  We instill 7 ml of HEPES-buffered Hanks balanced salt
solution (without Ca or Mg), and flush the instillate in and out a total
of three times.  The instillate is transferred to a sterile centrifuge
tube.  The procedure is repeated a minimum of 3 times and the cells are
pooled.  We normally obtain about 3 x 10^6 macrophages with about 95%
viability.

Macrophages can be isolated by adherance to a glass plate or by flow
cytometry.

We have maintained primary cultures in RPMI 1640 supplemented with 10%
heat inactivated fetal calf serum for 1 to 2 weeks. (We generally also
supplement the medium with Penn/Strep).