Mem non essential amino acids

HCT-116 cells (DSMZ, Braunschweig, Germany) were grown in McCoy’s 5A medium (Biowest) containing 10% fetal bovine serum (FBS) (PAA Laboratories, Pasching, Austria). Caco-2 cells (ECACC, Salisbury, UK) were grown in MEM medium (Biowest, Nuaillé, France) containing 15% FBS (PAA Laboratories) and MEM non-essential amino acids (Biowest). Culture media were supplemented with 2 mM glutamine (Biowest) and Zell Shield antibiotics (Minerva Biolabs, Berlin, Germany). Cells were maintained in a humidified atmosphere at 37 °C and 5 °C CO₂.

Human colon adenocarcinoma Caco-2 cell line (DSMZ ACC 169) was cultured in Dulbecco′s Modified Eagle′s Medium (DMEM, Gibco™) supplemented with 20% (v/v) Fetal Bovine Serum (FBS, Gibco™), 1% (v/v) MEM non-essential amino acids (Biowest), and 1% (v/v) Pen-Strep-Amp B (AB, Biological Industries, Cromwell, CT, USA), hereafter referred to as complete DMEM. Cells were incubated at 37 °C in humidified conditions with 5% CO₂, the medium was renewed biweekly, and cells were subcultivated upon reaching 80% confluency. Cell passage numbers ranging between 6 and 30 were used for these assays.
The heterogenous plurihormonal cell line STC-1 (intestinal secretin tumor, CRL-3254 ™) was cultured with Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum (FBS, Gibco™), and 1% AB (v/v). Cells were grown and maintained at 5% CO₂ at 37 °C with medium renewal every 2 to 3 days. Cells were passaged when cell confluency reached 70%, and the passage number used for this assay was between 6 and 34. All mammalian cell work was conducted using sterile technique on LAF benches.

FaDu cells were purchased to the American Type Culture Collection, and the HNSCC cell line SCC38 derived from a primary tumor (T2N0M0) was kindly provided by Dr. R. Grenman (Department of Otolaryngology, University Central Hospital, Turku, Finland) [52 ]. Primary cancer-associated fibroblasts (CAFs) were obtained from minced tumor tissue of surgically resected HNSCC at the Hospital Universitario Central de Asturias. Normal dermal fibroblasts (NF) were obtained from the dermis of human neonatal foreskin, by enzymatic cell disaggregation as described [53 ]. Cell line authentication was performed by DNA (STR) profiling at the SCT Core Facilities (University of Oviedo, Asturias, Spain). All cell lines were tested periodically for mycoplasma contamination by PCR to specifically amplify a conserved region of the mycoplasma 16S ribosomal RNA gene (Biotools Detection kit, Madrid, Spain).
HNSCC cells and fibroblasts were grown in DMEM (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA), 100 U/mL penicillin (Biowest, Nuaillé, France), 200 mg/mL streptomycin (Biowest, Nuaillé, France), 2 mM L-glutamine, 20 mM HEPES (pH 7.3) (Biowest, Nuaillé, France), and 100 mM MEM non-essential amino acids (Biowest, Nuaillé, France).