Singlequot kit

The human virus-transformed bronchial epithelial cell line BEAS-2B was purchased from ATCC and cultured in BEGM media (Lonza), supplemented with a SingleQuot kit (Lonza) and antibiotic-antimycotic solution (Invitrogen) in a 5% CO₂ incubator. All culture containers were pre-coated with 30 μg/ml PureCol (Advanced BioMatrix).

The CuFi-1 and NuLi-1 cell lines were a generous gift of A. Klingelhuts, Pkarp, and J. Zbaner, University of Iowa, Iowa City [27 (link)], and were cultured as previously described [25 (link)]. Briefly, the cells were cultured as monolayer in a humidified atmosphere at 37°C and 5% CO₂ in flasks precoated with collagen (collagen IV from human placenta, Sigma-Aldrich) in bronchial epithelial growth medium (BEGM by Lonza; singleQuot Kit Lonza).

HUVECs (3.0 × 10⁵ cells/ml) were seeded in 24-well plates in endothelial cell growth basal medium-2 (EBM-2; Lonza) containing EGM-2 (SingleQuot kit; Lonza). When HUVECs had formed a confluent cell monolayer, cells were starved in serum-free EMB-2 for 24 h. Cell monolayers were scratched with 200-μl pipette tips and carefully rinsed with PBS to create uncovered areas in the center of the cultured wells. Conditioned media (CM-L, CM-R), EBM-2 supplemented with EGM-2 (positive control), and serum-free EBM-2 (negative control) were added to culture plates. Images were captured immediately following media replacement and at 12 h and 36 h with a microscope (Axio Vert; Zeiss). The images were analyzed, and wound areas were measured using an optimized plugin for ImageJ to automatically recognize the wound healing size. The percentages of wound closure were calculated using the following equation: $$\text{Wound closure}\left(\%\right)=100\left({\text{A}}_{\text{T}=0}-{\text{A}}_{\text{T}=\mathrm{\Delta }\text{t}}\right)/{\text{A}}_{\text{T}=0}$$ A_T=0 is the initial wound area (μm²) and A_T=Δt is the wound area after 24 h or 36 h of the initial scratch (μm²).

NHBE cells (lot no. 0000442483; Lonza Walkersville, Walkersville, MD) were plated at 3,500 cells/cm² in culture flasks of bronchial epithelial cell growth medium supplemented with the SingleQuot^® kit (BEGM medium, CC-3170; Lonza Walkersville, Walkersville, MD) and cultured at 37 °C in a 5% CO₂ incubator. The medium was changed every 48–72 h and cells were grown to confluence for six days.