Coon s modified ham s f 12 medium

Coon’s modified Ham’s F-12 medium and human insulin were purchased from Sigma Chemical Co. (St Louis, MO, USA). Penicillin/streptomycin solution, neurobasal medium, and B27 were purchased from Gibco-Thermo Fisher Scientific (Waltham, MA, USA), and bFGF was purchased from R&D Systems (Minneapolis, MN, USA). LPS (Escherichia. coli O55:B5) was obtained from MilliporeSigma (St. Louis, MO, USA). The following antibodies were purchased: anti Bcl-2 (#SC-7382) from Santa Cruz Biotechnology (Dallas, TX, USA); anti β-tubulin type III (TUJ1) (#801202) from BioLegend (San Diego, CA, USA); and anti-calnexin (#ADI-SPA-860-F) from Enzo Life Sciences (Farmingdale, NY, USA). Fluorescein-(DTAF)-conjugated streptavidin (#016-010-084) was purchased from Jackson Immuno Research (West Grove, PA, USA). All other chemicals were of analytical grade.

The bronchial epithelial cell line CFBE41o- with and without stable co-expression of F508del-CFTR or wild type CFTR was cultured with MEM medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The generation of CFBE41o- cells co-expressing F508del-CFTR and the HS-YFP was previously described (Sondo et al., 2011 (link)). The FRT cells expressing F508del-CFTR and HS-YFP (Pedemonte et al., 2010 (link)) were cultured in Coon’s modified Ham’s F-12 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

The following cell cultures were employed: CFBE41o- and Fischer rat thyroid (FRT) cells with stable expression of F508del-CFTR (F508del-CBFE41o- and F508del-FRT, respectively) as well as CFBE41o- cells expressing a wild-type CFTR (wt-CFBE41o-) [29 (link)]. CFBE41o- cells were cultured in minimal essential medium (MEM), and FRT cells were cultured in Coon’s modified Ham’s F-12 medium (Sigma-Aldrich, St. Luis, MO, USA), both supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (0.1 mg/mL of penicillin and streptomycin), at 37 °C and 5% CO₂ in 75 cm² flasks. Stable expression is maintained by adding puromycin (0.5 μg/mL or 2 μg/mL for wt-CBFE41o- or F508del- CBFE41o-, respectively) and 0.6 mg/mL zeocin for FRT cell line to the complete culture medium. CF and non-CF primary bronchial cells were from the University of Pittsburgh CF center cell culture core facility. They were cultured in flasks and maintained at air–liquid interface (ALI) until being fully differentiated as polarized cells for experiments.