Aecg medium

Human nasal epithelial cells (HNEpCs), which were established by PromoCell GmbH, were suspended in an AECG medium (PromoCell GmbH) at a concentration of 1 x 10⁵ cells/ml, and were used as a target. To examine the influence of IL-4 on NO production from HNEpCs, 1 x 10⁵ cells (1.0 ml) were introduced in triplicate into 24-well culture plates containing various concentrations (0.5 to 30.0 ng/ml) of IL-4 to make a final volume of 2.0 ml. After 24 to 72 hours, the culture supernatants were collected and stored at -40°C until use. To examine the influence of quercetin and leflunomide on NO production from HNEpCs after IL-4 stimulation, 1 x 10⁵ cells (1.0 ml) were introduced in triplicate into 24-well culture plates containing various concentrations of either quercetin or leflunomide. The cells were then stimulated with 10.0 ng/ml of IL-4 for 48 hours in a solution with a total volume of 2.0 ml. The culture supernatants were collected and stored as mentioned above. To examine the influence of quercetin on the transcription factor, STAT6, activation, and iNOS mRNA expression in HNEpCs, 1 x 10⁵ cells were cultured in a similar manner for 12 and 24 hours, respectively [29 (link)]. In all experiments, quercetin and leflunomide were added to cell cultures 2 hours before stimulation.

Healthy bronchial specimen were obtained from patients undergoing elective pulmonary resection (Table S1). The patient’s informed consent was obtained before surgery and the studies were approved by the ethics committee of the medical faculty of the Otto-von-Guericke-University Magdeburg (vote 163/17, 16 October 2017). hbEC were isolated by cutting the tissue in 1 mm² pieces and plating them with the mucosal side on a petri dish. The tissue was supplied either with AECG medium (PromoCell, Heidelberg, Germany) or PC Ex+ medium (STEMCELL Technologies, Saint Égrève, France) during epithelial cell outgrowth and expansion. For isolation of hbFb, the tissue was cut in 1 mm² pieces and digested overnight in 5 mL DMEM (Gibco, Waltham, MA, USA), 0.75 mL collagenase B (Thermo Fisher Scientific, Waltham, MA, USA) and 50 µL antibiotic, antimycotic solution (Sigma-Aldrich, Darmstadt, Germany). The suspension was filtered first through a 100 µm and then a 40 µm filter and seeded in cell culture flasks, with fibroblast growth medium 2 containing 2% fetal calf serum (PromoCell). hbECs and hbFbs were passaged when a confluency of 80% was reached. The media were changed every two to three days. All cells were maintained under standard culture conditions in a humidified incubator containing 5% CO₂ at 37 °C.

HNEpCs, purchased from PromoCell GmbH, were suspended in AECG medium (PromoCell GmbH) at a concentration of 5 × 10⁵ cells/mL and used as target cells. The HNEpCs (5 × 10⁵ cells/mL) were stimulated with 12.5–100.0 μM H₂O₂ for 24 h in a final volume of 2.0 mL. To examine the influence of quercetin on TRX production, the HNEpCs (5 × 10⁵ cells/mL) were stimulated with 50.0 μM H₂O₂ in the presence of 0.1–10.0 nM quercetin for 24 h in a final volume of 2.0 mL. To examine TRX mRNA expression, the cells were stimulated with 50.0 μM H₂O₂ in the presence of 0.1–10.0 nM quercetin for 12 h. Quercetin was added to the cell cultures 2 h before H₂O₂ stimulation.