Lung Primary Fibroblasts Isolation Protocol

Procedure:
  1. Sacrifice mice.
  2. Rinse mice with 70% EtOH.
  3. Use a set sterile tools to cut out off the chest.
  4. Keep lungs in cool HBSS or serum free media.
  5. Cut the lungs into small pieces by using razor blade.
  6. Put the lungs into a falcon and add 4 mL of digestion buffer for 10 days old mice or 2 mL for new born mice. 37C for 90 minutes, mix gently every 30 minutes.
  7. Drain the pieces of lungs with 70 um filter and wash with 15 mL of cool serum physio or PBS (w/o Ca2+) with 0.05M EDTA (add 1/10 Vol 0.5M EDTA) to keep cells separated.
  8. Spin at 1500rpm for 5 min.
  9. Wash the pellet with 10 ml medium.
  10. Spin again at 1500rpm for 5 min.
  11. Resuspend the pellet in 1 mL medium.
  12. Plate the cells 100 mm plate containing 5ml media for 10days old lungs. Plate the cells in 60 mm plate containing 2ml media for new born to 3 days old lungs.
  13. Wash and change the media after 24 hours. You might have to wash 2x to 4x depending the number of blood cells in the dish.
20mL digestion buffer: 11 mL lysis buffer  + 4 mL CA + 5 mL Dispase (final [] 0.1% collagenase, 2.4U/ml dispase, 2 mM CaCl2 and 10mM HEPES) for cell culture. NS Serum physio.
 
Lysis buffer: NaCl 150 mM, Hepes 10 mM, CaCl2 2mM. Filter sterilize.
                   NaCl FW 58.44, CaCl2 dihydrate FW 147, Hepes stock is 1M
 
To 500 ml water add:
4.383 grams NaCl
0.147 grams CaCl2 dihydrate
5 ml 1M Hepes
 
Dispase II 
Dispase II. Roche. # 04942078001. 538$ for 5 bottles
 
1 gram in bottle
0.96 U/mg
Initial stock 10 mg/ml – dissolve one bottle (1 gram) in 100 ml saline/CaCl2/hepes
This is 9.6 U/ml
Sterile filter
 
Make 2.5 ml or 5 ml or 10 ml aliquots and store frozen -80.
Stock solution is stable at 4C for 2wks; stable frozen up to 2 months.
 
Collagenase A 
Collagenase A. 1g. Roche. # 11088785103. 195$
 
1 gram in bottle
use at 0.05% to 0.1% for lung single cell suspension
 
Make stock at 0.5% = 1 gram / 200 ml saline/CaCl2/Hepes
Sterile filter
 
Make 1.0 ml aliquots
 
Make Dispase/collagenase for lung digestion
Dispase final 2.4U/ml
Collagenase A final 0.1% or 0.05%
 
+ Single Edge Prep Razor blades. Fisher Scientific. # 12-640-18. 34.14$
+ Cell Stainer 40uM nylon. BD biosciences. # 352340 brand BD Falcon. 118.98$
+ MEM 1x. Fisher Scientific. # MT 17-305_CV. 63.6 $ 6*500ml