Glutamine

MCF-7 cells (sex: female, ECACC, Cat. No. 86012803) and MCF7 cells stably expressing EGFR-EGFP (EGFR^-EGFP MCF7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (PAN Biotech), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (PAN Biotech), 10mM glutamine (PAN Biotech) and 1% Non-Essential Amino Acids (PAN Biotech) at 37°C with 5% CO₂. MCF10A (sex: female, ATCC-CRL 10317) were grown in DMEM/F12 media supplemented with 5% horse serum, 20ng/ml EGF (Sigma-Aldrich), 0.5μg/ml hydrocortisone (Sigma #H-0888), 100ng/ml cholera toxin (Sigma), 10μg/ml insulin (Sigma) and 1% glutamine. MCF7 and MCF10A cells were authenticated by Short Tandem Repeat (STR) analysis and did not contain DNA sequences from mouse, rat and hamster (Leibniz-Institut DSMZ). Cells were regularly tested for mycoplasma contamination using MycoAlert Mycoplasma detection kit (Lonza).

Female C57BL6 mice, 7 weeks of age, were purchased from Charles River and were acclimatized for 1 week under standard laboratory conditions. Immediately after sacrifice, femurs and tibias were removed and cleaned of connective tissue, the ends were cut, and the marrow was flushed with α-MEM (PAN Biotech, Dutsher) supplemented with 15% FCS (PAN Biotech), 2 mM of glutamine (PAN Biotech), 50 IU/ml of penicillin (PAN Biotech), and 50 μg/ml of streptomycin (PAN Biotech). Single-cell suspensions were prepared in α-MEM by drawing the cells several times through graded needles. Cell density was determined using a Malassez counting chamber. For all experiments, cells were plated at a density of 2 × 10⁶ cells/cm² and incubated at 37°C in 5% CO₂. After 24 hours, non-adherent cells were removed and the medium was changed every 3 days until cell confluence.
Adipogenesis was induced by DMEM (PAN Biotech) 10% FCS supplemented with 10 μg/ml insulin/0,5 μM dexamethasone/100 μM indomethacin/500 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Corporation) for 4 days and maintained in 10 μg/ml insulin/0,5 μM dexamethasone/5 μM pioglitazone (Sigma-Aldrich Corporation) for 10 days (mBM-Adi).

Collagenase digestion was carried out with minor modifications according to [16 (link)]. Vital tumor tissues were cut into small pieces of approximately 2 mm³ and washed two times with phosphate-buffered saline (PBS). Collagenase (2.5 mg/mL, Nordmark Biochemicals, Uetersen, Germany) was dissolved in 0.5 M tris(hydroxymethyl)aminomethane and 150 mM Calcium chloride. Collagenase digestion was carried out for 1.5–2 h at 37 °C with regular vortexing. Cells were washed with PBS and suspended in medium: DMEM/Hams F12 supplemented with 20% fetal calf serum (FCS), glutamine (2 mmol/L), and antibiotics (medium and antibiotics were from Pan Biotech, Aidenbach, Germany, FCS from Sigma–Aldrich, Darmstadt, Germany and glutamine from Biochrom, Berlin, Germany). Single cells were obtained by using a 100 µm cell strainer. Single cells and remaining tissue were separated into two different 6-wells. Medium was changed regularly. Growing cell cultures were seeded into 25 cm² flasks and the serum concentration was serially reduced to 10% (with exception of HNSCC48 P0 M1: final serum concentration 15%).

HaCaT keratinocytes were cultivated in RPMI 1640 cell culture medium containing 8% fetal bovine serum (Sigma-Aldrich, Germany), 2 mM glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin (PAN Biotech, Germany) at 37°C, 95% relative humidity, and 5% CO₂ [16 (link)]. Twenty-four hours prior to experiment, 1 × 10⁶ cells were seeded in 60 mm dishes (Sarstedt, Germany). As cold physical plasma source, the kINPen 09 (neoplas tools, Germany) was utilized. This plasma jet consists of a central pin-type electrode that ignited a plasma by applying a voltage of 2–6 kV at a frequency of around 1 MHz. Argon (Air Liquide, France) was used as feed gas (3 standard liters per minute). For all experiments, an indirect treatment regimen was chosen to assure homogeneity of the treatment and it was achieved by exposing 5 ml of RPMI w/ all supplements to the plasma effluent at a distance of 9 mm using an automated xyz-table. The treated liquid was transferred immediately to the prepared cells.

The human lymphoma cell line Jurkat (ACC282) and the human lymphoblast cell line TK6 (ATCC CRL-8015) were used. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; PAN-Biotech, Aidenbach, Germany) or Roswell Park Memorial Institute (RPMI) 1640 medium (PAN-Biotech, Aidenbach, Germany), each containing 10% fetal bovine serum (Sigma-Aldrich-Chemie, Taufkirchen, Germany), 1% glutamine (PAN-Biotech), and 1% penicillin/streptomycin (PAN-Biotech, Aidenbach, Germany) at 37 °C and 5% CO₂. Before the treatment, the cells were seeded at 5 × 10⁴ in 100 µL DMEM into 96-well round bottom plates (Thermo Fisher Scientific, Dreieich, Germany). For a different set of experiments, 2.5 × 10⁵ cells in 500 µL of RPMI medium were seeded into flat-bottom 24-well plates (Eppendorf, Hamburg, Germany).