• Blunt curved dissection tweezers
• Dulbecco's Modified Eagle Medium: Nutrient Mixture F12 (DMEM/F12) (Gibco, 11320033)
• Penicillin-Streptomycin (Gibco, 15140122)
• Dulbecco’s phosphate-buffered saline (DPBS) (Gibco, 14190144)
• 24-well plate (Costar, 3524)
• Collagen (Sigma-Aldrich, C8919-20ml)
• Fibronectin (Merck, F2006-1MG)
• Bovine Serum Albumin (BSA) (Sigma-Aldrich, A7030-500G)
• Hanks’ Balanced Salt Solution (HBSS) (Gibco, 14025092)
• 70µm strainers (Miltenyi, 130-098-458)
• 40µm strainers (Miltenyi, 130-098-462)
• Dispase II (Sigma-Aldrich, D4693-1G)
• DNase I (Roche, 10104159001)
• ACK Lysing Buffer (Gibco, A1049201)
• Anti-CD45 MACS beads (Miltenyi, 130-052-301)
• Anti-CD31 MACS beads (Miltenyi, 130-097-418)
• Anti-EpCAM MACS beads (Miltenyi, 130-105-958)
• LS MACS columns (Miltenyi, 130-042-401)
• MS MACS columns (Miltenyi, 130-042-201)
• Fc block CD16/CD32 (BioLegend, 101301)
• OctoMACS separator (Miltenyi, 130-042-109)
• QuadroMACS separator (Miltenyi, 130-091-051)
• Airway Epithelial Cell Growth Medium (Promocell, C-21060)
• DMH-1 (StemCell, 73632)
• A 83-01 (StemCell, 72022)
• Y-27632 (StemCell, 72302)
• CHIR9901 (StemCell, 72052)
• Trypan Blue (Gibco, 15250061)
• Stericup filter unit (Milipore, S2GPT01RE)
MACS buffer wash - DPBS (no Mg2+ and Ca2+) + 0.5% BSA + 2mM EDTA + 1% Pen/Strep
Well-plate coating solution - 3% collagen, 1% fibronectin and 1% BSA in HBSS. Distribute at least 0.2ml per 1cm2. Incubate for at least 4-8h at 37°C, then keep up to 5 weeks at 4C⁰ covered in film.
Airway epithelial growth media – 1µM A 83-01, 0.2µM DMH-1, 5µM Y27632 ROCK, 0.5µM CHIR9901 and 1% v/v Penicillin/Streptomycin in supplemented Promocell Airway Epithelial Cell Growth Medium. Keep up to 7 days at 4°C and protected from light.
Digestion solution - 2mg/ml of Dispase II resuspended in sterile DMEM/F12 + 0.1mg/ml DNase I + 1% v/v Penicillin/Streptomycin filtered through 0.22µm filter. Aliquot to Bijous (5ml) and freeze down. Thaw aliquots one hour before the start of the protocol.
Dispase wash - 50µg/ml of DNaseI with 1% v/v Penicillin/Streptomycin in DMEM/F12.