Coll. A/Elastase/Dispase Enzyme Mix (1.5 mL --> save 0.5 mL of coll./elastase/dispase mix in separate 1.5 mL tube. )
90 µL Collagenase A 100 mg/mL – 6 mg/mL final (Sigma, 10103578001)
150 µL elastase 43 u/mL - 4.3 u/mL final (Worthington, LS002292)
150 µL Dispase 90 u/mL – 9 u/mL final (Worthington, LS02100)
7.5 µL 1 M CaCl2 – 5 mM final
7.5 µL DNAse (125 U/mL) (Applichem, A3778)
B. Lich enzyme mix (1 mL)
899 µL DPBS (no Ca, no Mg) (Thermo Fisher, 14190144)
100 µL Bacillus Licheniformis 100 mg/mL - 10 mg/mL final conc. (Sigma, P5380)
Preparing stock aliquots of reagents:
Enzyme stocks are made up in DPBS. I make up 100 mg/mL stock of Collagenase A and Bacillus Licheniformis. For elastase, I prepare 43 u/mL stock, and for dispase a 90 u/mL stock. Enzymes mixes are aliquoted in 150 µL (elastase, depase) or 100 µL (Collagenase A, Bacillus Licheniformis) and stored at -80 ºC. For the CaCl2, I prepare a 1 M stock and autoclave. The DNAse is diluted in DPBS, aliquoted and stored at -80 ºC.
Red Blood Cell Lysis Buffer (Sigma, R7757) - store at RT
Trypan blue solution, 0.4% (ThermoFisher, 15250061)
(Optional) Dead Cell Removal Kit:
EasySep dead cell removal (Annexin V) kit (cat. #17899)
EasySep Magnet (cat. #18000)
Required Equipment & Consumables:
Centrifuges for 1.5 mL and 15 mL conicals (MLS)
Pipettes and pipet tips (MLS)
15, 50 ml Conicals (MLS)
1.5 mL tubes (MLS)
30 µM filters - Miltenyi (130-098-458)
Petri dishes (MLS)
Razor blades (MLS)
Ice bucket w/ice (MLS)
Hemocytometers - InCyto Neubauer Improved (DHC-NO1-5)
The protocol workflow is as follows:
A. Isolate lung
B. First layer
C. Second layer
D. Preparing cells for Chromium