Insulin like growth factor

Rat type II alveolar cells (AEC II/ RLE-6TN) obtained from the America Tissue Type Collection (ATTC, Manassas, VA, USA, cat. CRL-2300) were cultured using Ham’s F12 medium (Biochrom GmbH, Berlin, Germany, cat. FG0815) containing 2 mM L-glutamine and supplemented with 10% Fetal Bovine Serum (PanBiotech GmbH, Aidenbach, Germany, cat. 0522D), 0.01 mg/mL bovine pituitary extract (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA, cat. 13028-014), 0.005 mg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA, cat. I-6634), 2.5 ng/mL insulin-like growth factor (Sigma-Aldrich, cat. I-3769), 0.00125 mg/mL transferrin (Sigma-Aldrich, cat. I-1147), and 2.5 ng/mL epidermal growth factor (Sigma-Aldrich, cat. I-9644). Cells were grown at 37 °C in a 5% CO₂ humidified atmosphere. The medium was changed every 2–3 days and the cells were subcultured when at 80–90% confluence. AEC II were sown in complete Ham’s F12 medium at a density of 2.5 × 10⁴ cells/cm² and cultured for 24 h prior to treatment.

RLE-6TN cells (lot no. 59111690), a rat lung epithelial cell line with characteristics of alveolar type II cells, were purchased from the American Type Culture Collection (Rockville, MD, USA). All experiments using this cell line were performed within 4 months after resuscitation. RLE-6TN cells were grown in Ham's F12 medium containing 2 mM L-glutamine (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 10 μg/ml bovine pituitary extract (PromoCell, Heidelberg, Germany), 5 μg/ml insulin (Gibco), 2.5 ng/ml insulin-like growth factor (Sigma-Aldrich, St. Louis, MO, USA), 1.25 μg/ml transferrin (Gibco), and 2.5 ng/ml epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA) as we described previously (Hagiyama et al., 2015 (link)). H₂O₂ (Wako Pure Chemical Industries, Osaka, Japan) was added to the semiconfluent culture of RLE-6TN cells at a concentration of 440 μM, according to the procedures published previously (Kim et al., 2014 (link)). After 4 or 8 h, H₂O₂ was removed by medium replacement, and the cultures were continued for 2 or 3 days. In another experiment, elastase (porcine pancreas; Worthington, Lakewood, NJ, USA) was added at a concentration of 1 unit/ml to the semiconfluent culture of RLE-6TN cells grown in the Ham's F12 medium same as described above except lacking fetal bovine serum. After 1 h, cells were subjected to Western blot analyses.

After accurate cell counting, 5 × 10³ WB cells in 25 μl of DMEM/F12 medium (5%FBS, 50 ng/ml epidermal growth factor (Sigma) and 1 μg/ml insulin‐like growth factor(Sigma)) mixed with 25 μl Matrigel(BD Pharmingen) were added to a low attachment 96 wellplate (SorfaBio, Beijing, China). The 96 well plate was placed in the incubator for an hour and then added with 100 μl medium. After 3 days, 200 μl culture medium was replenished and added with different doses of capsaicin. The medium was changed every 2 days. Spheres were observed and captured.