Dulbecco s modified minimum essential medium

The 16HBE cell culture was obtained from Millipore Sigma (St. Louis, MO). Cells were passaged weekly for no more than 17 weeks before returning to frozen cell stocks. This correlates to actual serial passages 47–64 from the establishment of the original immortalized cell line. No discernible changes in morphology or physiology were observed within this window of passages. After reaching confluence, cells were trypsinized (0.25% trypsin and 2.2mM EDTA) (Corning Cellgro, Manassas, VA) and then passaged on a weekly basis by seeding 3.0 x 106 cells per Falcon 150 cm2 culture flask with 50ml of Dulbecco’s Modified Minimum Essential Medium (Corning Cellgro, Manassas, VA), supplemented with 2mM L-Glutamine (Corning Cellgro, Manassas, VA), 10% fetal bovine serum (Seradigm, VMR, Inc., Radnor, PA), 1% non-essential amino acids (Corning Cellgro, Manassas, VA), and 1mM sodium pyruvate (Corning Cellgro, Manassas, VA). Cultures were incubated at 37°C in 95% air/5% CO₂ atmosphere. Confluent cell density was approximately 3.3x10⁵ cells/cm².

The 16HBE cell culture was obtained from Millipore Sigma (St. Louis, MO) and used for 15 weeks before returning to frozen cell stocks. After reaching confluence, cells were trypsinized (0.25% trypsin and 2.21 mM EDTA) (Corning Cellgro, Manassas, VA) and then passaged on a weekly basis. Cells were seeded at 1.5 × 10⁶ cells per Falcon 75-cm² culture flask with 25 ml Dulbecco’s Modified Minimum Essential Medium (Corning Cellgro, Manassas, VA), supplemented with 2 mM l-Glutamine (Corning Cellgro, Manassas, VA), 10% fetal bovine serum (Seradigm, VMR, Inc., Radnor, PA), 1% non-essential amino acids (Corning Cellgro, Manassas, VA), and 1 mM sodium pyruvate (Corning Cellgro, Manassas, VA). Cultures were incubated at 37°C in 95% air/5% CO₂ atmosphere.