Newborn calf serum ncs

DF-1 cells (a spontaneously immortalized chicken embryo fibroblast (CEF) cell line, ATCC catalog no. CRL-12203), primary CEF cells (obtained from specific-pathogen-free 11-day-old eggs; MSD, Salamanca, Spain), and HeLa cells (immortalized human epithelial cervix adenocarcinoma cells, ATCC^® CCL-2) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (FCS) (Gibco-Life Technologies, Carlsbad, CA, USA) for DF-1 and CEF cells or 10% newborn calf serum (NCS) (Sigma-Aldrich, St. Louis, MO, USA) for HeLa cells. Cell cultures were maintained at 37 °C in a humidified incubator containing 5% CO₂. Cell lines were infected with viruses, and after 1 h of adsorption, the virus inoculum was removed and DMEM-2% FCS or DMEM-2% NCS was added to the cell cultures.

HEK-293T cells (ATCC catalog no. CRL-3216), HeLa cells (ATCC CCL-2), DF-1 cells (ATCC CRL-12203) and Vero-E6 cells (ATCC CRL-1586) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 100 U/mL penicillin/100 µg/mL streptomycin (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 0.1 mM non-essential amino acids (Sigma-Aldrich), 0.5 μg/mL amphotericin B (Fungizone; Gibco-Life Technologies, Waltham, MA, USA) and 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich) for HEK-293T, DF-1 and Vero-E6 cells or 10% heat-inactivated newborn calf serum (NCS; Sigma-Aldrich) for HeLa cells. Cell lines were maintained in a humidified air, 5% CO₂ atmosphere at 37°C.

To evaluate the protective effects of HA derivatives in an oxidative stress-induced model of oral mucosal injury in vitro, immortalized normal human oral keratinocytes (OKF6) cultures were used as a cellular model of oral mucosa. OKF6 [35 (link)] is a benign human oral keratinocyte cell line which expresses hTERT and bypasses a p16^INK4a cell cycle control mechanism and derived from the floor of the mouth, provided by Oral Health Cooperative Research Centre (OHCRC), The University of Melbourne, Australia (underwent karyotype analysis prior to experimentation,VCGS Cytogenetics Laboratory, Royal Children’s Hospital, Parkville, VIC). Cells were cultured as discribed elswhere [67 (link)]. Briefly, OKF6 cells were cultured as adherent cultures in 100-mm tissue culture-treated plates and grown in keratinocyte serum-free medium (K-SFM) (#17005-042, Thermo Fisher Scientific) supplemented with 25 μg/mL pituitary bovine extract (PBE), 0.2 ng/mL epidermal growth factor (EGF), 1% (v/v) Newborn Calf Serum (NCS) (#N4637, Sigma-Aldrich), 100 IU/ml penicillin, and 100 μg/ml streptomycin (#P4333, Sigma-Aldrich, Castle Hill, NSW, Australia), and containing 0.4 mM calcium chloride CaCl₂. Cells were grown in a humidified atmosphere at standard conditions (5% CO₂ at 37 °C).