Premium grade fbs

A stock culture of INT 407 cells (ATCC CCL 6) was cultured in Minimal Essential Media (MEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Premium Grade FBS, 100% US Origin, VWR, Product No. 97068-085) and 1 mM sodium pyruvate (Corning Inc., Manassas, VA, USA) at 37 °C in a 5% CO₂ incubator. Adherence assays were performed as outlined elsewhere [30 (link)], with one modification—the bacteria were suspended in MEM without FBS. All assays in this study were performed at a multiplicity of infection (MOI) ranging between 50 and 500 to one to ensure reproducibility and repeated a minimum of three times. Regardless of the MOI, the trends of the isolates relative to one another were the same.

The human bronchial epithelial cell (HBEC6-KT) line was cultured in Keratinocyte Serum-Free Medium 1X (Gibco) according to the manufacturers directions and supplemented with P/S (VWR) at 1X^{70 (link)}. Human airway epithelial cell line Calu-3 (ATCC HTB-55) were cultured in Minimum Essential Medium Alpha Medium (Corning) and supplemented with Premium Grade FBS (VWR) at 10%, HEPES (Corning) at 1X, and P/S (VWR) at 1X. The following chemical reagents were dissolved in DMSO, cAMP elevating agents Forskolin (Cayman Chemical) and Isoproterenol (Cayman Chemical), ABCC4 inhibitors MK-571 (Cayman Chemical) and Ceefourin 1 (Abcam), PDE inhibitors Roflumilast (Cayman Chemical) and Rolipram (Cayman Chemical), and CFTR Inhibitor (Selleck Chemicals).