Singlequot kit supplements and growth factors

NHBE cell line (Lonza) from one nonsmoking donor was cultured in a bronchial epithelium growth medium (BEBM medium with SingleQuot Kit Supplements and Growth Factors, Lonza) on 75 cm² until 85% confluent. Subsequently, the cells were passaged (3 × 10⁵ cells on 1.2 cm⁵) onto collagen-coated transwell inserts in a 12-well culture plate (Costar, Corning) and were cultivated until confluent. Afterwards, the basolateral chamber medium was switched to the air-liquid interface (ALI) (1 : 1, BEBM : DMEM, 3.5 g/L D-glucose with SingleQuots), supplemented with 100 nM retinoic acid (Sigma-Aldrich). The apical surface of the cell culture was then exposed to air. The medium was replaced three times per week. Any mucus that has appeared on the top of cells was systematically removed. Cilia were observed 3–4 weeks posttransition to ALI. Differentiated cells were scratched by P200 Gilson pipette tip. Cell debris was removed, and a fresh medium was added to the basolateral chamber.

HEK293T cells (293T, GenTarget) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Corning Cellgro) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals), 100 U/mL penicillin, and 100 µg/mL streptomycin (Life Technologies). Brain microvascular endothelial cells (BMECs) were obtained from American Type Culture Collection (ATCC) and expanded in endothelial cell growth medium (Lonza) supplemented with SingleQuot Kit Supplements and growth factors (Lonza). All cultures were maintained at 37°C with 5% CO₂.