Stable glutamine

CHO DP-12 clone #1933 ATCC® CRL-12444^TM [34 ] was cultured in CDM4CHO medium (Hyclone, UT, USA) supplemented with 6 mM stable glutamine (Biowest LLC, MO, USA), 0.002 mg/ml Humulin N (Eli Lilly, IN, USA) and 200 nM methotrexate (Pfizer, NY, USA), at 37ºC in a 5% CO₂ atmosphere, in a humidified incubator. Cells were seeded in duplicate at 0.5 x 10⁶ cells/ml and a viability higher than 95%, in 35 ml medium in 250 ml Erlenmeyer flasks, at 60 rpm (Bellco Glass, NJ, USA). Cell concentration and viability were recorded every 24 h by cell counting in a Neubauer chamber, using the trypan blue dye exclusion method.

Primary cultures of osteoblasts were set up from trabecular bone fragments taken during hip arthroplasty surgery. Specifically, the fragments were first washed in phosphate buffered saline (PBS) and then incubated at 37°C with 1 mg/mL porcine pancreatic trypsin ≥60 U/mg (SERVA Electrophoresis GmbH Heidelberg, DE) diluted in PBS. Subsequently, bone fragments were subjected to repeated digestions with 2.5 mg/mL collagenase NB 4G Proved grade ≥0.18 U/mg (SERVA Electrophoresis GmbH, Heidelberg, DE) diluted in PBS with calcium and magnesium. After digestions completion, the supernatant was collected and centrifuged at 340 RCF for 10 min. Cells were seeded into a 24-well plate at a density of 2 × 10⁴ cells/well and maintained in DMEM-F12 (Biowest SAS, Nuaillé, France) supplemented with 10% fetal bovine serum (FBS) (Biowest SAS, Nuaillé, France) growth medium, 100 Units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, United States) and 2 mmol/L stable glutamine (Biowest SAS, Nuaillé, France) in an incubator at 37°C, 5% CO₂ until reaching confluence. The medium was changed every 3–4 days. During RPM exposure, the growth medium was supplemented with 50 μg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO, United States) and 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, United States).

The human colorectal adenocarcinoma cell line HT29 was purchased from the American Type Culture Collection (ATCC) and maintained in RPMI1640 with stable glutamine (Biowest) enriched with 10% fetal bovine serum (FBS)(Biowest), 1% penicillin/streptomycin (Welgene), and 25 mM HEPES (Shimakyu Co., Ltd., Japan), 25 mM NaHCO₃ (Shimakyu Co., Ltd.,) at 37°C with 5% CO₂. Isolated MNL lymphocytes were cultured in RPMI 1640 with L-glutamine and sodium bicarbonate (Sigma), 10% FBS (Biowest), 1% penicillin/streptomycin (Welgene), and 0.05 mM 2-β-mercaptoethanol (Sigma) at 37°C in the presence of 5% CO₂. For western blot analysis, Gos/Fos, inulin, and β-glucan were treated in the presence of 0, 0.25, 0.5, 1, 2, 5 % w/v, respectively for 12 h.

DMS114 cells (human small cell lung cancer, ATCC
CRL-2066) obtained from American Type Culture Collection (Manassas,
VA) were cultured in the Waymouth’s MB 752/1 medium from Gibco
(Waltham, MA). MCF7 cells (human adenocarcinoma, ATCC HTB-22) obtained
from American Type Culture Collection were cultured in DMEM high glucose
with stable glutamine and sodium pyruvate from Biowest (France). The
MCF7 cells stably expressing FGFR1 (MCF7-R1) were generated by transfection
of pcDNA3-FGFR1 using DOTAP (Roche Diagnostics). Clones were selected
with 1 mg/mL G-418 and chosen based on their receptor expression level
analyzed by immunofluorescence and immunoblotting. MCF7-R1 cells were
cultured in DMEM high glucose (Biowest) with 50 μg/mL gentamicin
sulfate (Thermo Fisher Scientific), stable glutamine, and sodium pyruvate
(Biowest).

The mouse chondrogenic cell line ATDC-5 was kindly gifted by Dr. Oreste Gualillo (University of Santiago de Compostela, La Coruña, Spain). Cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM)–Ham’s F-12 medium (Biowest, Nuaillé, France) supplemented with 5% FBS (Gibco, UK), 10 μg/mL human transferrin (Sigma-Aldrich, St. Louis, USA), 3 × 10⁻⁸ M sodium selenite (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin-streptomycin-amphotericin B (Biowest, Nuaillé, France), and 1% stable glutamine (Biowest, Nuaillé, France) in a humidified atmosphere of 5% CO₂ at 37 °C.
The viability of cells was assessed using the Blue Cell Viability Assay (Abnova, Taipei, Taiwan). In brief, cells (10⁴ cells/well) were cultured in 96-well plates and treated with onion extracts (OE) (1.9–62.5 µg/mL) for 24 h at 37 °C. After that, the reagent was added in accordance with the manufacturer’s instructions (10%; incubation of 4 h at 37 °C and 5% CO₂). Metabolically active cells were able to reduce the dye, and the fluorescence generated was calculated in a microplate reader (Varioskan Lux, Thermo Fisher, Waltham, MA, USA) at 530 nm excitation and 590 nm emission wavelengths. Viability percentages were determined by linear interpolation of data considering control samples (non-treated cells) as 100% of viability.

Cell lines CHO DP-12 clones #1933 (CRL-12444) and 1934 (CRL-12445) ATCC® (Manassas, VA, the USA) [26] were used in this work. Both cell lines were adapted to grow in suspension, and both produce humanized anti-IL-8 MAbs in different proportions. Both cell lines were cultured in CDM4CHO medium (GE Healthcare, Chicago, IL, the USA) supplemented with 6 mM stable glutamine (Biowest LLC, Nuaillé, France), 0.002 mg/ml Humulin N (Eli Lilly, Indianapolis, IN, the USA) and 200 nM methotrexate (Pfizer, New York, NY, the USA), at 37ºC in a 5% CO₂ atmosphere in a humidified incubator (Model NU-5500, Nuaire, Plymouth, MN, the USA). The culture medium was supplemented with 20% (v/v) PowerFeed A (BE02-044Q, Sartorius AG, Göttingen, Germany).
The inoculum was expanded in 75 cm² T-flasks at an orbital agitation of 60 rpm (Cat. 7644–10115 Bellco Glass Inc., Vineland, NJ., the USA), and cells were seeded at 0.30 × 10⁶ cells/ml in duplicate in 25 cm² T-flasks with a filled volume of 8 ml. Cell concentration and viability were recorded by cell counting in a Neubauer chamber using the trypan blue dye exclusion method (Model AE2000, Motic, Hong Kong, China) [27].