Cholera toxin solution

Murine BECs were isolated by cold enzymatic digestion of murine bronchi or tracheas as described previously [49 , 50 (link)]. Single-cell suspensions from mice murine bronchi or tracheas were cultured in 12-well plates that were coated with collagen I (50 μg/ml, BD Biosciences, Franklin Lakes, New Jersey, USA) at 3.5 × 10⁵ cells/ml of MTEC (HyClone, USA) proliferation media containing RPMI-1640 medium (Gibco-ThermoFisher Scientific, USA), 10% heat-inactivated FBS, retinoic acid stock B (10 mmol/l, Sigma-Aldrich), insulin solution (6.25 mg/l, Sigma, USA), epidermal growth factor solution (50 ng/ml, BD Biosciences), bovine pituitary extract (25 mg/l; Sigma-Aldrich), transferrin solution (6.25 mg/l, Sigma-Aldrich) and cholera toxin solution (4.2 mg/l, Sigma-Aldrich). The submerged MTEC cultures were incubated at 37 °C in a humidified incubator containing 95% air and 5% CO₂. After 6 h, the supernatant and non-adherent cells were discarded. The adherent cells were allowed to differentiate for 7–10 days by replacing the proliferation medium with MTEC basal medium containing Nu-seum (2%, BD Biosciences) and retinoic acid (10 mmol/l, Sigma–Aldrich). BECs were centrifuged and stained with cytokeratin-specific monoclonal antibody and DAPI.

Murine BECs were obtained by cold enzymatic digestion of murine bronchi or tracheas. Single cell suspensions from mice were cultured in 12-well plates that were coated with collagen I (50 µg/ml; BD Medical Technology, Franklin Lakes, New Jersey, U.S.A) at 3.5  ×  10⁵ cells/ml of MTEC proliferation media containing RPMI-1640 medium (Gibco-Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A), 10% heat-inactivated FBS (Gibco-Thermo Fisher Scientific), retinoic acid stock B (10 mmol/l; Sigma–Aldrich, St. Louis, Missouri, U.S.A), insulin solution (6.25 mg/l; Sigma–Aldrich), epidermal growth factor solution (50 ng/ml; BD Medical Technology), bovine pituitary extract (25 mg/l; Sigma–Aldrich), transferrin solution (6.25 mg/l; Sigma–Aldrich), and cholera toxin solution (4.2 mg/l; Sigma–Aldrich). The submerged MTEC cultures were incubated at 37°C in a humidified incubator containing 95% air and 5% CO₂. After 72 h, the supernatant and non-adherent cells were discarded. The adherent cells were allowed to differentiate for 10–14 days by replacing the proliferation medium with MTEC basal medium containing Nu-serum (2%; BD Medical Technology) and retinoic acid (10 mmol/l; Sigma–Aldrich).