L glutammine

Caco-2 cells from passage 5 to 20 were cultured in 25 cm² surface area flasks (Corning Inc. Life Sciences, New York, NY, USA) using Dulbecco’s modified Eagle’s medium (DMEM, Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 20% fetal bovine serum (FBS, Gibco, ThermoFisher Scientific, Waltham, MA, USA), 1% L-glutammine 200 mM (Gibco, ThermoFisher Scientific, Waltham, MA, USA), 1% penicillin–streptomycin 10,000 U/mL (Gibco, ThermoFisher Scientific, Waltham, MA, USA) and maintained at 37 °C under 5% CO₂. Cells were seeded at 104 cells/cm² in 96-well plates (Corning Inc. Life Sciences, New York, NY, USA) for cytotoxicity assay.

PBMCs were loaded with Carboxyfluorescein succinimidyl ester (CFSE, Life Technologies). Briefly, 1x10⁶ cells/ml were resuspended in PBS (Euroclone) with 1% FBS (Gibco BRL) and loaded with 1μM CFSE for 20 min at 37°C. After washing, cells (at a concentration of 5x10⁶ cells/ml) were stimulated with 0.35 μM of TLR9 agonist CpG-B ODN2006 (Hycult Biotech) in complete medium for 5 days at 37°C. Complete medium was prepared as follows: RPMI-1640 (Euroclone), 10% heat inactivated fetal bovine serum (FBS, Hyclone Laboratories), 1% L-Glutammine (Gibco BRL); 1% Penicillin/Streptomicin 100X (Euroclone), 1% sodium pyruvate (Gibco BRL).

MDA-MB-231 breast cancer cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Life Technologies, Paisley, SCT, UK). BT-549 and BT-474 breast cancer cell lines were cultured in Roswell Park MEMorial Institute 1640 Medium (RPMI 1640, Gibco, Life Technologies, Paisley, UK). MCF-7 cells were cultured in Modified Eagle’s Medium (MEM, Gibco, Life Technologies, Grand Island, NE, USA). Cell lines were purchased from the American Type Culture Collection (ATCC) and cultured in humidified atmosphere containing 5% CO₂ at 37 °C. The media were supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and were used after addition of 50 U/mL penicillin, 50 μg/mL streptomycin, 2 nM L-glutammine (all from Gibco, Life Technologies, Paisley, UK). The cells were cultured as previously described [38 (link)].

Immediately after withdrawal, ACD-anticoagulated blood was centrifuged at 100 g, 10 minutes at room temperature. The platelet rich-plasma was completely removed and the remaining blood was mixed with RPMI (Lonza, 1640) containing 0,1% L-glutammine (Thermo Fisher), 0.5% Pen-strep (Thermo Fisher), 20% FCS and 0.38% sodium citrate (Sigma) to restore the initial volume of blood in the tubes. Mononuclear cells were isolated by Ficoll-Paque Plus (GE Healthcare, vWR Int.) density centrifugation at 600 g, 20 minutes at room temperature with no brake. Cells were washed with PBS containing 0, 1% glucose (Sigma), 0, 5% BSA (Sigma) and 5 mM EDTA and centrifuged at 830 g for 10 minutes at 4 °C with brake. Cells were further washed twice in PBS without EDTA and centrifuged at 350 g for 10 minutes at 4 °C with brake. Mononuclear cells resuspended in RPMI supplemented with 10% autologous serum, 0, 1% L-glutammine and 0.5% Pen-strep were then plated (100.000 monocytes/100 µl) in a 48 Well Cell Culture Plate and incubated at 37 °C. After 2 hours cells were washed 3 times with PBS in order to remove non-adherent lymphocytes; adherent monocytes were then cultured in the same medium over 7 days at 37 °C (5% CO₂). Medium was not replaced throughout the culture period.

Primary cortical neurons were prepared as described before [52 (link)]. Briefly, cortices from mice embryos at embryonic day 16.5 were dissociated mechanically, and neurons were resuspended in Neurobasal™ medium supplemented with B27 (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) containing 30 U/mL Penicillin, 30 mg/mL Streptomycin (Sigma-Aldrich, Milan, Italy), 0.75 mM Glutamax (Gibco™, Thermo Fisher Scientific), and 0.75 mM L-Glutammine (Gibco™, Thermo Fisher Scientific). Depending on the experiment, neurons were seeded 80.000 cells/1.9 cm² on 0.1 mg/mL Poly-D-Lysine-coated glass coverslip (Sigma-Aldrich, for transfection with miRNA expression vectors) or 300.000 cells/10 cm² on 0.02 mg/mL Poly-D-Lysine-coated 6-multiwell plates (for transfection with miRNA mimics) and maintained at 37 °C under a 5% CO₂ humid atmosphere. Three days after seeding, half of the medium was replaced with 24 h astrocyte-conditioned medium. Then, half of the medium was changed every seven days for up to a maximum of four weeks.