Allprep dna rna protein isolation kit

Primary human tracheal epithelial cells isolated from the surface epithelium of human trachea (HTEpC, lot n° 454Z019.11, PromoCell GmbH, Heidelberg, Germany) were cultured at 37 °C (5% CO₂) in 60x15mm standard tissue culture dishes (cat. 351,007, Corning, NY, USA) with Airway Epithelial Cell Growth Medium (cat. C-21060, PromoCell) prepared with SupplementMix (cat. C-39165, PromoCell) and Gibco antibiotic-antimycotic solution (cat. 15,240,096, ThermoFisher Scientific, MA, USA).
Once the cells reached 10⁵ cells per plate (30–40% confluency), HTEpC were washed 3 times with sterile 1× PBS pH 7.4 (cat. 10,010,072, ThermoFisher), and fresh media was added to each plate. Cells were exposed to 2 mW/cm² (at a distance of 1.5 cm from the light source) of NB-UVA for 20 min based on previously validated ideal UVA irradiation levels [6 (link)]. The temperature of the medium/cells during exposure to NB-UVA was checked every 5 min during the experiments using an infrared thermometer (Lasergrip 774, ETEKCITY, CA, USA). Unexposed cells were used as controls. After 24 h the supernatants were collected, and cell were washed 3 times with sterile 1× PBS, pH 7.4. Following the removal of any remaining PBS, cells were lysed in the plate using 1 mL of RTL buffer from an AllPrep DNA/RNA/Protein isolation kit (Qiagen, Hilden, Germany). Experiments were performed in triplicate.

Supernatants collected from UVA-exposed and control HTEpC from the previous experiment were transferred to a new 60 × 15 mm tissue culture dish containing 10⁵ naïve HTEpC (i.e. cells that were never exposed to UVA). Before receiving the supernatant from UVA-exposed or control cells, the naïve HTEpC were washed 3× with sterile 1× PBS, pH 7.4. The PBS was completely removed, and 4 mL of the supernatant collected from UVA-exposed or control HTEpC were added to the naïve cells. After 24 h of incubation, the cells were washed 3×, and were then lysed in the plate using 1 mL of RTL buffer from an AllPrep DNA/RNA/Protein isolation kit (Qiagen). Experiments were performed in triplicate.