1) Sacrifice the mice and bleed them by aortic section.
2) Excise the lungs from thoracic cavity and place the lungs on a sterile petri dish containing HBSS (Invitrogen).
3) Mince the lung tissue into 1 cubic centimeter pieces and transfer the tissue to a tube containing 15 ml dissociation medium (Dissociation medium: RPMI 1640 (Invitrogen), 5% fetal calf serum (FCS) (Invitrogen), collagenase type I (150 U/ml) (Sigma)
4) Seal the tube and place the tube on a rotor at 37C for 2 hours.
5) Pour the lung tissue on to a mesh and collect the liquid into a beaker underneath.
6) Place the wire mesh screen on a sterile petri dish and rinse the tissue 2 times with a pipette using 3 ml of cold HBSS.
7) Push the lung tissue through the mesh screen using the rubber end of a sterile syringe plunger. Work under a sterile hood.
8) Rinse the plunger and the mesh with HBSS and repeat the process 5 times.
9) Transfer the contents of the petri dish to a conical tube and let the tube site on ice until the particulate matter settles. This usually takes about 4-5 minutes.
10) Transfer the liquid into a centrifuge tube and leave aggregates of lung tissue behind.
11) Centrifuge the cell suspension at 300 g for 15 min.
12) Remove the liquid and resuspend the cell pellet in 10 ml of HBSS.
13) Centrifuge the cell suspension in HBSS again and remove the liquid and resuspend the cell pellets again and repeat this process 5 times and finally resuspend the pellet in 1-5 ml RPMI 1640 containing 5% FCS.
14) Count the cells.