Immortalized Gingival Keratinocyte Culturing

Immortalized human oral gingival keratinocytes, IHGK (HPV-16GM), were purchased from Applied Biological Materials (ABM, cat# T0717, Richmond, CA, USA), and maintained as we previously described [58 (link)]. Briefly, gingival epithelial cells (GECs) were cultured and maintained in Keratinocyte serum-free medium (KSFM, Gibco, Gaithersburg, MO, USA) suplemented with 30 μg/mL of bovine pituitary extract (Gibco), 0.2 ng/mL of human recombinant epidermal growth factor (Gibco), 100 U/mL of penicillin and 100 μg/mL of streptomycin (Gibco). All cells were maintained at 37 °C in a humidified incubator containing 5% CO₂. Cell counts were performed using Trypan Blue (Sigma-Aldrich, MO, USA) exclusion for seeding only viable cells for the experiments.