Lhc 9 media

BEAS-2B cells (SV40-immortalized human airway bronchial epithelial cell line) and A549 cells (human lung adenocarcinoma epithelial cell line) were purchased from the American Culture of Tissue Collection and grown in complete growth medium (RPMI 1640 and DMEM supplemented with heat-inactivated 10% FBS and 1% L-glutamine, respectively) at 37^oC/5% CO₂. Before use, cells were starved in minimum medium (RPMI 1640 or DMEM supplemented with 1% FBS and 1% L-glutamine), and cell culture supernatants were harvested at different time points. To eliminate the contamination of supernatant by free-floating cells, the supernatant was centrifuged and the upper half of the medium was taken as the sample.
Human primary bronchial epithelial cells obtained from three subjects without COPD and three subjects with COPD were cultured as monolayers in LHC-9 media (Invitrogen) on collagen (1% w/v)-coated plates as previously reported.⁴⁰ (link) Cells were extracted from lung tissue of patients undergoing lung resection at the Royal Brompton Hospital. All subjects gave informed written consent and the study was approved by the National Research Ethics Service London-Chelsea Research Ethics committee, study No. 09/H0801/85. All cells were serum starved 16 hours before stimulation. Cells were stimulated with 3% cigarette-smoke-conditioned media prepared as previously reported.⁴¹ (link)

BEAS-2B cells (catalogue number CRL-9609) were purchased directly from American Type Culture Collection (Rockville, MD). Keratinocyte serum-free medium (K-SFM), bovine pituitary extract (BPE), and recombinant human epidermal growth factor (EGF) were purchased from Invitrogen (Paisley, UK). BEAS-2B cells were cultured in K-SFM containing 50 μg/ml BPE and 5 ng/ml EGF at 37°C in a humidified atmosphere comprising 5% (v/v) CO₂ in air. Cells were cultured as monolayers in growth factor-free media for 24 h prior to stimulation. Human primary airway epithelial cells were cultured as monolayers from bronchial brushings from both male and female subjects (Table 1) and cultured in LHC-9 media (Invitrogen, Paisley, UK) in collagen (1% w/v) coated flasks. Samples were obtained from non-smokers, smokers and patients with COPD. Smokers had a smoking history of at least 10 pack-years and COPD patients were stable and fulfilled the American Thoracic Society criteria [26 (link)]. The subjects were matched for age and smokers and COPD patients for smoking history (Table 1). All subjects gave informed written consent and the study was approved by the NRES London-Chelsea Research Ethics committee, study number 09/H0801/85.

Human primary bronchial epithelial cells were cultured as monolayers in LHC-9 media (Invitrogen, Paisley, UK) on collagen (1% wt/vol)-coated plates. Cells were extracted from lung tissue from patients undergoing lung resection surgery at the Royal Brompton Hospital. All subjects gave informed written consent, and the study was approved by the National Research Ethics Service London-Chelsea Research Ethics Committee (Study No. 09/H0801/85). The BEAS-2B cell line (SV40-immortalized human airway bronchial epithelial cell line) were purchased from the American Culture of Tissue Collection and were maintained in complete growth medium (RPMI 1640 supplemented with heat-inactivated 10% FBS and 1% l-glutamine) at 37°C/5% CO₂. Before use, cells were starved for 24 h in minimum medium (RPMI 1640 supplemented with 1% FBS and 1% l-glutamine). N-acetyl cysteine (10 mM) was also given 10 min before CSE treatment.