CFTR-W1282X Cell Culture Protocol

FRT cell lines were cultured in F12 Coon’s modification media (Sigma, F6636) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PenStrep). 16hBEge-W1282X cell lines were obtained from the Cystic Fibrosis Foundation (CFF) and cultured according to their instructions in minimum essential medium (MEM) media (32 (link), 72 (link)). Single-cell clones of the original CFF16hBEge-W1282X cell line were created to select for high-resistance clonal cell lines. One clonal cell line, CFF16hBEge-W1282X-SCC:3F2, was selected for analysis. Primary hBE cells isolated from CF patients homozygous for CFTR-W1282X (patient code HBEU10014) and compound heterozygous for CFTR-W1282X and CFTR-F508del (patient code HBEND12112) were also obtained from the CFF. For functional analysis, cells were differentiated by plating on Costar 24-well high-throughput screening filter plates (0.4 μM pore size, Polyester, Corning, catalog No. CLS3397). FRT and 16hBE cells were grown in a liquid/liquid interface (180 μL apical/700 μL basolateral) in a 37 °C incubator with 90% humidity and 5% CO₂ for 1 wk. Primary hBE cells were differentiated in an air/liquid interface for 5 wk. Media was replaced three times a week.