Primary lung fibroblasts cultures

Single cell suspension from lungs

Materials and supplies required:

• Collagenase 4 (Worthington #LS004189)
• Dispase 2 
• DNAse (Worthington #LS002007)
• Serum free medium
• PBS*1
• Red blood cell lysis buffer (0.8g NH4Cl, 0.1g KHCO3 in 100 ml DDW – filtered in 0.2uM, kept in 4⁰). can also use Pharmalyse instead (BD #555899)
• 10% FCS medium
• Cell Strainer 70 mm (Falcon #2350)

Preparations: 

• Mix Collagenase solution (immediately before use):

• If sterility is required, filter the solution through 0.2 mm filter in the bio-hood after mixing.
• Bring box with ice
• Prepare sterile surgery tools.
• Warm water bath to 37⁰

Procedure:

1. Euthanize mice, and remove lungs into the small glass jar (on ice). Do the same for all the mice.
2. Mince the tissue using curved scissors and forceps in the digestion jar, on ice.
3. Add 20ml collagenase mix solution, insert magnet and immediately transfer to water bath.
4. Place the glass container in a 37^oC water bath for 40 minutes on top of the stir apparatus. 
   1. While in bath - coat plate with collagen (per 6 plate well 1 ml acetic acid with 12.5 microliter collagen, half an hour in incubator, then rinse twice with PBS and the plate is ready).
5. After 40 mins, remove from bath and stop reaction by adding 25-30 ml of DMEM 10% FCS.
6. Strain the digested mixture through a 70mm cell strainer placed on top of a 50 ml conical tube.
7. Spin the conical tube with cells in the Tissue Culture centrifuge for five minutes at 1500 rpm. 
8. Aspirate the supernatant using a glass pipette attached to a vacuum, without disturbing the cell pellet.
9. Red blood cells lysis: re-suspend the cells in 5-10 ml of RBC lysis buffer.
10. Let sit in RT for 5 min. To stop reaction, add 30 ml of DMEM+10% FBS (PBS is also fine, the solution is not an enzyme and to stop the lysis diluting it is enough).
11. Spin the conical tube with cells in the Tissue Culture centrifuge for five minutes at 1500 rpm.
12. Aspirate the supernatant using a glass pipette attached to a vacuum, without disturbing the cell pellet.
13. Re-suspend in growing medium and plate on collagen coated 6 well plate (approx. one mouse per well)
14. Cells usually take 4-7 days to recover.

[1] Collagenase 4 works at the efficiency of 50-200 units/ml. the stock contains ­>160 units per mg, 20 mg in 20 ml SFM à 160 units/ml. when using less or more medium à use less or more collagenase 4 accordingly

[2] Dispase2 works at the efficiency of 0.6-2.4 units/ml. the stock contains 0.96 units per mg, 20 mg are in 20 ml SFM à 0.96 units/ml. when using less or more medium à use less or more Dispase accordingly.