Ham s f 12k media

P. aeruginosa lab strains PAO-1 (ATCC 15692) and ATCC27853, as well as clinical CRS-isolates P11006, 2338, and L3847 (obtained from Drs. N. Cohen and L. Chandler, Philadelphia VA Medical Center) (73 (link)) were grown in LB media (Gibco). For anti-bacterial assays, P. aeruginosa were grown to OD 0.1 in Luria broth (LB) and resuspended in 50% saline containing 1 mM HEPES and 0.5 mM glucose, pH 6.5. Nasal ALIs were washed 24 hrs prior with antibiotic-free Ham’s F12K media (ThermoFisher Scientific) on the basolateral side. 30 uL of bacteria solution was placed on the apical side of the ALI for 10 min, followed by aspiration of bulk ASL fluid. After 2 hrs at 37°C, remaining bacteria were removed from the ALI culture by washing followed by life-dead staining with SYTO9 (live) and propidium iodide (dead) with BacLight Bacterial Viability Kit (ThermoFisher Scientific; cat # L7012). Control experiments were performed similarly using transwell filters with no cells and bacteria in solution ± 10 µg/ml colistin sulfate. Green (live)/red (dead) ratio was quantified in a Spark 10M (Tecan, Mannedorf, Switzerland) at 485 nm excitation and 530 nm and 620 nm emission. CFUs counting was done by taking aliquots of bacteria saline solution from similar experiments without the live/dead stain, diluting with saline as indicated, and spotting on LB agar plates.

The human normal urethral epithelial cell line, SVHUC1, and two human BLCA cell lines, including T24, and UMUC3, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). SVHUC1 cells were maintained in Ham's F-12K Media (Invitrogen, USA). T24 and UMUC3 cells were incubated in Dulbecco’s Modified Eagle Medium (Invitrogen, USA). Those media were supplemented with 10% FBS and 1% antibiotics, and all of the cells were cultured in a humidified atmosphere with 5% CO2 at 37° C.

The human NSCLC cell lines A549 and H23 were purchased from ATCC. A549 cells were cultured in Ham’s F12K media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA), H23 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, Mo., USA) supplemented with 10% (vol/vol) FBS, HEK293 cells were purchased from ATCC and grown were cultured in DMEM media containing 10% (vol/vol) FBS. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.

Human lung adenocarcinoma A549 cells (at passage #91) were purchased from European Collection of Cell Cultures (ECCC, distributor Sigma Aldrich). A549 cells were seeded at a density of 7000 cells/cm² in T-75 culture flasks. They were cultured in Ham’s F12K media (Gibco), supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich) and 1% Penicillin-Streptomycin (BioReagent, Sigma Aldrich).
Prior to the nanowire exposure, cells were seeded at 1.2 x 10⁴ cells/cm² in a 35 mm Petri dish with a collagen-coated glass coverslip at the bottom. Cells were let to adhere and adapt to the environment for 24 h.
Collagen-coated glass coverslips were prepared as follows: coverslips were sterilized under UV light for 10 min and incubated in 100 mg/mL collagen water solution (Corning) for 24 hours at room temperature in a sterile laminar flow hood. Collagen coated-coverslips were washed once in Ham’s F-12K media, and placed on the bottom of the 35 mm Petri dish.