PBMC were isolated from blood using a Ficoll-Paque PLUS (GE Amersham) gradient [66 (link)] and maintained in RPMI (Invitrogen). PBMC were used for functional studies.
Primary human normal airway tracheobronchial epithelial cells from de-identified donors (NHBE) (sex: male and female) were provided by Lonza (Walkersville, MD) and grown in BEM media supplemented with the BEGM bullet kit (Lonza). NHBE were used for functional studies, and for generation of polarized human airway epithelial cultures (HAE). To generate HAE, NHBE from individual donors were expanded on plastic to generate passage 1 cells, which were subsequently plated (5x104 cells/well) on rat-tail collagen type 1-coated permeable transwell membrane supports (6.5mm; Corning Inc). HAE cultures were grown in B-ALI medium supplemented with inducer (Lonza Inc.) at each media change with provision of an air-liquid interface for approximately 6 weeks to form differentiated, polarized cultures that resemble in vivo pseudostratified mucociliary epithelium.
Primary human normal airway tracheobronchial epithelial cells from de-identified donors (NHBE) (sex: male and female) were provided by Lonza (Walkersville, MD) and grown in BEM media supplemented with the BEGM bullet kit (Lonza). NHBE were used for functional studies, and for generation of polarized human airway epithelial cultures (HAE). To generate HAE, NHBE from individual donors were expanded on plastic to generate passage 1 cells, which were subsequently plated (5x104 cells/well) on rat-tail collagen type 1-coated permeable transwell membrane supports (6.5mm; Corning Inc). HAE cultures were grown in B-ALI medium supplemented with inducer (Lonza Inc.) at each media change with provision of an air-liquid interface for approximately 6 weeks to form differentiated, polarized cultures that resemble in vivo pseudostratified mucociliary epithelium.
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