Streptomycin pen strep

Human lung cancer cell lines (A549 and H1299) and normal lung cells (BEAS‐2B) were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% foetal bovine serum (FBS, Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin (pen/strep, Sigma). The cells were cultured at 37°C in the presence of 5% CO₂.

HS-5 conditioned medium (CM) was generated by incubating confluent HS-5 (ATCC, Manassas, VA) in complete RPMI medium consisting of RPMI 1640 (Sigma-Aldrich, St. Louis, MO), 10% FBS (Thermo Fisher), 2 mM L-glutamine (Sigma-Aldrich), 100 IU/mL Penicillin and 0.1 mg/mL Streptomycin (Pen-Strep, Sigma-Aldrich), for 72 h. The supernatant was filtered through 0.2 µm and frozen at −80 °C until use.

To obtain conditioned medium (CM) generated from hAMTCs, freshly isolated hAMTCs were cultured for 3 days in 24‐well plates (Corning, NY, USA) at a density of 3 × 10⁵ cells/well in 0.5 ml Roswell Park Memorial Institute (RPMI) complete medium, composed of RPMI 1640 medium (Sigma‐Aldrich, St Louis, MO, USA) supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Sigma‐Aldrich), 2 mm l‐glutamine (Sigma‐Aldrich) and 100 U/ml penicillin and 100 mg/ml streptomycin (pen–strep, herein referred to as P/S; both from Sigma‐Aldrich).
To obtain CM without prostaglandins (CM – PG), hAMTCs were cultured as described above in the presence of 10 μm indomethacin (Sigma‐Aldrich), a cyclooxygenase inhibitor. PGE2 quantification in CM and CM – PG was obtained using a Prostaglandin E2 EIA Kit (Cayman Chemical Co., Ann Arbor, MI, USA), according to the manufacturer's instructions. Absorbance was measured at 405 nm using a microplate reader.
At the end of the culture period, CM and CM – PG were collected, centrifuged at 300 × g, filtered through a 0.8 μm sterile filter (Sartorius Stedim, Florence, Italy) and kept frozen at −80 °C until use.
To obtain results that were least influenced by single donor variability and more representative of soluble factors released by hAMTCs, 10 pools, each containing CM from three to six different cell preparations, were used.