Glutamine

Primary cells were obtained from mucosal melanoma tumour tissue. The tumour tissue was cut into little pieces and dried on culture dishes upside down for 30 minutes before medium was added. After three to four days, the tissue was removed and adhered mucosal melanoma cells were cultured. The mucosal melanoma primary cells TU-MM1–TU-MM4, as well as the melanoma cell lines SK-MEL 30, IGR-37, MEL-JUSO, COLO-849 and SK-MEL 3 (DSMZ, Braunschweig, Germany) were cultured under standard conditions (37°C, 5% CO₂, fully humidified atmosphere) in Dulbecco's Modified Eagle's Medium or Roswell Park Memorial Institute 1640 medium (TU-MM3, TU-MM4, SK-MEL3), supplemented with 10% foetal calf serum (PAA Laboratories, Cölbe, Germany), 1% penicillin-streptomycin and 1% glutamine (all from Biochrom, Berlin, Germany). Normal human epidermal melanocytes (NHEM) were cultured in Melanocyte Medium 2 (PromoCell, Heidelberg, Germany). Cells were treated with 8 μM cisplatin (Teva, Ulm, Germany) for 24 hours before protein or RNA was isolated.

Tumor tissue from neuroblastoma patients was obtained from residual material after pathological analysis. Informed written consent was obtained from the parents and the protocol was approved by the local IRB (892007V). Histological diagnosis was confirmed by the Institute of Pathology, University of Tübingen. Tumor tissue was disrupted mechanically and placed in 2 mL DMEM medium containing 1 g/L glucose (LG-DMEM, Lonza, Basel, Switzerland), supplemented with 5% (v/v) human fresh frozen plasma (FFP), 10⁷/mL platelets (both University of Tübingen blood donor center), 80 IU/mL heparin sulfate (Medunasal, Isemhagen, Germany), 100 IU/mL penicillin and 100 mg/mL streptomycin (Biochrom, Berlin, Germany), 2 mM glutamine (Biochrom, Berlin, Germany) and incubated at 37 °C with 10% CO₂. T-MSC colonies appeared after 7–9 days. Non-adherent cells were removed, and adherent cells were detached using trypsin (Lonza, Basel, Switzerland) when confluent [17 (link)].
Specimens of BM-MSC were derived from excess material of standard bone marrow biopsies in children treated for leukemia (IRB approval 241/2005V). MSC cultures were established as reported earlier [33 (link)].

ND7/23 cells (Sigma-Aldrich, St. Louis, MO, USA) were grown on glass coverslips coated in poly-D-lysine, which were placed in 35 mm Petri dishes containing Dulbecco’s Modified Eagle Medium (DMEM; Biochrom GmbH) with 5% (v/v) fetal bovine serum (FBS superior; Biochrom, Berlin Germany), glutamine (Biochrom, Berlin Germany) and 100 µg/ml penicillin/streptomycin (Biochrom, Berlin Germany). Moreover, the growth media contained 1 µM all-trans Retinal. To transiently transfect cells, 6 µl FuGENE HD transfection reagent (Promega, Madison, WI) was incubated with 2 µg of vector DNA in 250 µl DMEM for 15 minutes and added to the cells two days prior to measurements.

Primary squamous carcinoma cell lines (University of Turku-Squamous Cell Carcinoma (UT-SCC)) have been established previously in the Grenman lab²⁶ and FaDu cells were from ATCC/LGC (Wesel, Germany). All cell lines were newly authenticated using STR profiling or obtained from the original source within the last four years. Cells were cultured under standard conditions (37 °C, 5% CO₂) in Dulbecco’s Modified Eagle’s Medium (DMEM) with Sodium Pyruvate (Sigma, Taufkirchen, Germany), supplemented with 10% (v/v) fetal calf serum and glutamine (both from Biochrom, Berlin, Germany).

The Cal27 and HN cell lines were obtained from DSMZ (Braunschweig, Germany), and UD-SCC-4 and UD-SCC-5 cells were obtained from the University of Düsseldorf (clinic for otolaryngology, Düsseldorf, Germany) (Tab. 3) [35 (link)]. The cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Invitrogen, Darmstadt, Germany) containing 10% fetal calf serum (FBS) (Biochrom, Berlin Germany), 2 mM glutamine (Biochrom), 100 μg/ml streptomycin (Biochrom), and 100 U/ml penicillin (Biochrom), maintained at 37°C in an atmosphere of 5% CO₂, and grown to 70–90% confluence.