Isolation of E12.5 mouse embryonic lung progenitors
Sox9-IRES-GFP (Sox9GFP; JAX 030137) or Wild-Type (C57BL/6J; JAX 000664) mouse strains
Molecular grade Ethanol 100%
Sterile 60 mL syringe (BD, 309653)
Sterile 20 mL syringe (BD, 309661)
Membrane Filter 0.22µm (Millipore, GSWP04700)
DPBS (Thermofisher, 14190144)
50 mL Falcon Tubes (Corning, 352070)
15 mL Falcon Tubes (Corning, 352096)
10cm Tissue Culture Dish (Corning, 353003)
100µm cell strainer (Corning, 352360)
40µm cell strainer (Corning, 352340)
5ml round bottom tubes with cell strainer (Corning, 352235)
Sterile Low-bind 1.5 mL tubes (Eppendorf, 022431081)
Dissociation Buffer
•Collagenase I 100U/mL (Thermofisher, 17100017)
•Collagenase II 100U/mL (Thermofisher, 17101015)
•Dnase 100U/mL (Roche, 04716728001)
•Trypsin 50U/mL (Thermofisher, 27250018)
•Phenol red-free Leibovitz L-15 (Thermofisher, 21083027)
Collection Buffer
•Sterile 20% FBS (Hyclone, SH30071)
•Phenol red-free Leibovitz L-15 (Thermofisher, 21083027)
Suspension Buffer
•Sterile 2% FBS (Hyclone, SH30071)
•HEPES 25mM (Thermofisher, 15630080)
•EDTA 2mM (Thermofisher, 15575020)
•Phenol red-free Leibovitz L-15 (Thermofisher, 21083027)
•DAPI 0.1µg/mL (Sigma, D9542) (before FACS sorting only)
FACS Buffer
•BSA 0.2% (Sigma, A9418)
•Sterile 2% FBS (Hyclone, SH30071)
•Phenol red-free Leibovitz L-15 (Thermofisher, 21083027)
Post-Sorting Collection Buffer
•Suspension Buffer
•Sterile 20% FBS (Hyclone, SH30071)
•RNasin Ribonuclease Inhibitor 40U/µL (Promega, N2115)
•G418 50µg/mL (Thermofisher, 11811031)
Antibodies
•anti-Cdh1 e660 (eBioscience, 50-3249-82)
•anti-Pecam1/CD31 e450 (eBioscience, 480311)
•anti-Ptprc/CD45 e450 (eBioscience, 480451)
•anti-Ter119 e450 (eBioscience, 485921)
Primary Sox9+ lung progenitor cell culture, passaging and cryopreservation
15 mL Falcon Tubes (Corning, 352096)
24 well Plates (Corning, 353047)
Advanced DMEM/F12 (Thermofisher, 12634010)
Growth Factor Reduced, Phenol Red-Free Matrigel 10mg/mL (Corning, 356231)
LPM3D Medium (store at 4°C, use before 10days)
•Advanced DMEM/F12 (Thermofisher, 12634010)
•Fgf10 50ng/mL (R&D, 6224-FG-025)
•Fgf9 50ng/mL (R&D, 7399-F9-025)
•Egf 50ng/mL (R&D, 2028-EG-200)
•CHIR99021 3µM (Tocris, 4423)
•BIRB796 1µM (Tocris, 5989)
•Y27632 10µM (Tocris, 1254)
•A8301 1µM (Tocris, 2939)
•Heparin 5µg/mL (Sigma, H3149)
•Insulin 10µg/mL (Roche, 11376497001)
•Transferrin 15µg/mL (Roche, 10652202001)
•PenStrep 1x (Thermofisher, 15140122)
•Glutamine 1x (Thermofisher, 25030081)
•Anti-Anti (optional) 1x (Thermofisher, 15240062)
•G418 (optional) 50µg/mL (Thermofisher, 11811031)
Dissociation Buffer for Passaging
•Dispase II (Thermofisher, 17105041) 0.5mg/mL
•Collagenase IV (Thermofisher, 17104019) 0.5mg/mL
•Papain (Worthington, LS003118) 0.5mg/mL
•Advanced DMEM/F12 (Thermofisher, 12634010)
Freezing Medium
•90% FBS (Hyclone, SH30071)
•10% DMSO (Sigma, C6295)
•Advanced DMEM/F12 (Thermofisher, 12634010)
CryoTube vials 1.8 mL (Thermo Scientific Nunc, 377267)