Dulbecco s modified eagle s

We cultured the gingival fibroblast cell lines in a t25 culture flask using a mixed medium of DMEM (Dulbecco’s Modified Eagles, Gibco, Waltham, MA, USA), 10% (v/v) fetal bovine serum (Invitrogen, Waltham, MA, USA.), Antibiotic-Antimycotic™ (Amphotericin B, Penicillin, Streptomycin) 1%, and GlutaMAX™ 1% (Invitrogen, Waltham, MA, USA). We then incubated this in an incubator (Shel Lab, Sheldon, Cornelius, OR, USA) that controls volume, with the following parameters: carbon dioxide 5%, temperature 37 °C, humidity 95%. We changed the cell culture every 2–3 days until the cells grew to 90% confluence.

For the purpose of this research, six-week-old male BALB/cByJNarl mice were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). Each cage housed two mice, and sterilized food and water were provided. The mice were kept at a constant temperature (22 ± 2 °C) and relative humidity (55 ± 5%) under a 12:12 h light:dark cycle. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the National Chiayi University (IACUC Approval No. 108028). All procedures were in line with the Guidelines for the Care and Use of Laboratory Animals by Taiwan’s Ministry of Health and Welfare.
The A549 human lung carcinoma cell line was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). The cells were grown in Dulbecco’s modified Eagle’s medium and supplemented with 5% fetal bovine serum, 50 IU/mL of penicillin, and 50 mg/mL of streptomycin (Gibco Laboratories, Grand Island, NY, USA) in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. The cells were routinely passaged by removing the medium and overlaying the cell monolayer with 0.25% trypsin and 0.1% EDTA.