Protocol for the isolation of macrophages from Lung tissue

1. Remove lung using sterile technique and lavage with sterile PBS
2. Place lung in culture media (i.e. RPMI 1640 with 10% FBS)
3. Place the lungs in 3ml of the same type of media used in step 2 in a small petri plate (35x10mm)
4. Place the lungs on a fine wire mesh screen (i.e. bar width 200um and open space 340um)
5. Push the lung through the screen with the plunger of a 10ml syringe into the petri dish from step 3
6. Wash the screen with 7ml of the same type of media used in step 2 into the petri dish from step 5
7. Transfer solution to centrifuge tubes and centrifuge at ~180rcf for 10 minutes
8. Discard supernatant
9. Resuspend pellet in 10ml of the same type of media used in step 2
10. Make a 1ml of a 1:10 dilution of the resuspended pellet from step 9 and count cells in hemocytometer or cell counter
11. Spin cells from step 9 at ~180rcf for 10 minutes
12. Discard supernatant
13. Resuspend cells at your working concentration in the same type of media as step 2
14. Incubate cells for 3 hours at 37oC in a %% CO₂ incubator
15. Remove media and discard
16. Replace media and return to incubator

Note: The macrophages should be attached after 3 hours incubation, however they may not be attached as well as macrophage cell lines like J774A.1, so refrain from vigorous removal of media or shaking.