Enzymatic digestion of the lungs
1. Dissect the mouse and extract the lung.
2. Chop the lung finely in 2% FCS in DMEM (200ml DMEM+ 4ml FCS).
3. Transfer the lung pieces into a 15ml tube.
4. Centrifuge 5min at 1,200rpm.
5. Transfer the lung pieces into a clean 15ml tube.
6. Resuspend in 3ml enzyme mix.
7. Incubate with rotations for 1h at 37οC.
8. Transfer the digested lung through a cell strainer (The cells are now floating in
the medium).
9. Centrifuge 5min at 1,200rpm. Red and white blood cells will pallet.
10. Red blood cell lyses by resuspending in 9ml DDW, pipette for 30-45 sec and
immediately add 1ml 10xPBS.
* Small DNA residues are sometimes visible, let them sink. Transfer the fluid into a new
tube.
11. Centrifuge 5min at 1,200rpm.
12. Resuspend in HBA buffer for FACS analysis.
Enzyme mix:
1.5 mg/ml Collagenase (Sigma C5138)
0.75 mg/ml Hyaluronidase (Sigma H3506)
0.1 mg/ml DNase (Sigma D5025)