Passage #3 KRT5+ cells were seeded (4.0 ×104/ insert) on purified collagen 1 (Advanced BioMatrix) coated Transwell polyester membrane inserts (6.5 mm diameter; 0.4 µm pore size; Corning). PneumoCult-Ex media (STEMCELL) containing manufacturer’s supplements, was added to apical and basal chambers and replaced 48-hourly. When confluent, the apical media was removed to promote mucociliary differentiation, and PneumoCult-ALI medium with manufactuer’s supplements (STEMCELL) and Primocin (InvivoGen, #ant-pm-1) was added to the basal chamber. From week 2 following air- exposure, mucus was gently washed from the apical surface with warmed PBS and stored for further analysis. Cells were maintained in ALI culture for 21 days, then inserts were fixed in 4% PFA (Electron Microscopy Sciences, #RT-15710) for immunofluorescence microscopy.
Pneumocult ali medium
Manufactured by STEMCELL
PneumoCult-ALI medium is a serum-free, animal component-free culture medium designed for the expansion and maintenance of human airway epithelial cells in an air-liquid interface culture system.
Automatically generated - may contain errors
2 protocols using pneumocult ali medium
1
Differentiation of KRT5+ Airway Epithelial Cells
2
Differentiation of Bronchial Stem Cells
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Differentiation of BSCs in the ALI was previously described (21 (link), 22 (link)). Briefly, BSCs were seeded on 0.4-μm Transwell membranes in Small Airway Epithelial Cell Growth Medium (PromoCell, catalog no. C-21070). After the BSCs reached confluency, the medium was removed from the top compartment, and Pneumocult-ALI medium (StemCell Technology, catalog no. 05001) was applied in the bottom compartment. The medium was changed every 2 days. For cytokine pretreatment, BSCs in Small Airway Epithelial Cell Growth Medium were treated for 7 days with IL6 alone (10 ng/ml; R&D Systems, catalog no. 206-IL-010/CF) or a cocktail containing IL6, IFN-γ (R&D Systems, catalog no. 285-IF-100/CF), and IFNλ2 (Sigma-Aldrich, catalog no. SRP3060), each cytokine at 5 and 10 ng/ml. After treatment, BSCs were passaged (splitting ratio, 1:4) followed by differentiation in the ALI. For treatment with S3I-201 (20 μM; Selleck Chemicals, catalog no. S1155), the ALI culture was treated from Day 0 to Day 7, fixed on Day 21 when epithelial differentiation was deemed complete, and processed for cryosection and antibody staining (18 (link), 27 (link)).
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