Required: - Liberase (Research Grade from Sigma-Aldrich Cat#05401119001) - Ammonium Chloride Solution (STEMCELL Cat#07800) - DNaseI   (STEMCELL Cat#07900) - RPMI 1640 Medium   (STEMCELL Cat#36750) - PBS+2%FBS (STEMCELL Cat#07905) - Sterile water - 70uM nylon mesh strainer (Falcon 352350)

1. Reconstitute Liberase (Research Grade from Sigma-Aldrich Cat#05401119001) with sterile water to a 2.5mg/mL concentration.  

2. Prepare Digestion Buffer by diluting Liberase 1 in 10 with RPMI 1640 Medium to a working concentration of 0.25 mg/mL. Add DNaseI Solution 1mg/mL to a final concentration of 0.25 mg/mL. (For example: Add 1mL of 2.5mg/ml reconstituted Liberase and 2.5mL of STI Cat#07900 DNaseI to 6.5mL of RPMI 1640, which will give enough for digesting 10 lungs)

3. Harvest mice lungs and temporarily place in 50mL tube containing PBS+2%FBS while collecting other lungs.

4. Carefully move collected lungs to an appropriately sized petri dish without medium and then use a razor blade, scalpel or scissors to prepare into homogeneous paste (< 1 mm in size).

5. Add Digestion Buffer (~1ml of digestion buffer per processed lung) to prepared paste and incubate at 37C on rocking platform for 30minutes with continuous agitation.

6. After incubation, pass the digested lungs through a 3mL syringe attached to a 20G needle to decrease viscosity. Repeat this processing with the syringe for a total of three times.

7. Place a 70uM nylon mesh strainer over a 50ml conical tube and then prime strainer with PBS+2%FBS.  

8. Add 3mL - 5mL of lungs per strainer. Mash through strainer with rubber end of a syringe plunger and rinse the filter thoroughly with PBS+2%FBS.

9. Place a new 70uM strainer over a new 50mL conical tube. Prime with PBS+2%FBS.  

10. Using a 10mL pipette, pass the contents of the original 50ml conical tube onto the new 50ml conical tube's strainer.  

11. Top up the new 50mL conical tube to 50ml with PBS+2%FBS.

12. Centrifuge at 300xg, for 6 minutes at room temperature with low brake setting.

13. Pipette off and discard supernatant. Next, add 3mL of Ammonium Chloride Solution (Cat#07850, STI) per lung to lyse for ~5 minutes at room temperature (for example, use 18mL of Ammonium Chloride Solution if originally started with 6 lungs).  

14.   After the time has elapsed, top up the 50mL conical tube to 50ml with PBS+2%FBS.

15. Centrifuge at 300xg, for 6 minutes at room temperature with low brake setting.

16. Aspirate supernatant, re-suspend cells in a small volume of PBS+2%FBS and execute a nucleated cell count. If desired, adjust to desired nucleated cell concentration and proceed to the EasySep immunomagnetic cell separation.