Glutamax

Human skeletal muscle cells (CC-2561 from Lonza) were cultured in DMEM/F-12, GlutaMAX™ (Life Technologies) supplemented with 20% FBS (Sigma-Aldrich) and 1% penicillin/streptomycin (Life Technologies) during proliferation. Differentiation was initiated when cells were 80% confluent by addition of differentiation media (DMEM/F-12, GlutaMAX™ supplemented with 2% FBS (Sigma-Aldrich) and 1% penicillin/streptomycin). Cells were differentiated for 5–7 days. For palmitate and TNFα treatment, the differentiated myotubes were added 0.5 μM palmitate for 48 h (on day 5–7 of differentiation) or 10 ng/ml TNFα for 24 h (on day 6–7 of differentiation).
Murine C2C12 myoblasts (CRL-1772 from ATCC) were cultured in DMEM (Life Technologies) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin/streptomycin (Life Technologies). Differentiation was initiated when cells were 80% confluent by addition of differentiation media (DMEM/F-12, GlutaMAX™ supplemented with 2% horse serum (Sigma-Aldrich) and 1% penicillin/streptomycin). Cells were differentiated for 5 days.
Insulin stimulation experiments for human skeletal muscle cells and C2C12 cells were performed by serum depriving differentiated myotubes for 4 h before stimulating with either 10 or 100 nmol/L insulin for 5 min.

Organotypic slice cultures were made as described previously (Kapfhammer and Gugger, 2012 (link)). Mice were decapitated at postnatal day 8, and their brains were aseptically dissected. The cerebellum was separated in ice-cold minimal essential medium (MEM) supplemented with 1% glutamax (Gibco, Invitrogen) and sagittal slices of 350 μm thickness were cut with a McIlwain tissue chopper under sterile conditions. Cerebellar slices were separated, transferred onto a permeable membrane (Millicell-CM, Millipore), and incubated with incubation medium (50% MEM, 25% Basal Medium Eagle, 25% horse serum, 1% glutamax, 0.65% glucose) or Neurobasal medium (97% Neurobasal medium, 2% B27, 1% glutamax) under 5% CO₂ at 37°C. The medium was refreshed every 2 or 3 days.

CMCs (10⁴ cells/cm²) derived from VII, XV, and XXXI subcultures were seeded on 6-well multiwell plates (Corning) previously conditioned with 2% gelatin (Merck) aqueous solution by a 2 h-incubation period at 37 °C. Cells were cultured with inductive medium composed of Neurobasal Medium (Life Technologies, Carlsbad, CA, USA), 2% B-27™ Supplement (Life Technologies), 20 ng/mL Epidermal Growth Factor (EGF) (Merck), 10 ng/mL basic Fibroblast Growth Factor (bFGF) (Merck), 1% glutamax (Merck), and 1% penicillin and streptomycin solution (Merck).
At 3 days from seeding, 50% of medium was replaced with fresh one, whereas, starting from 7 days, cells were cultured for 7 days with inductive medium composed of Neurobasal Medium, 2% B-27™ Supplement, 0.5 mM retinoic acid (RA) (Merck), 20 ng/mL Nerve Growth Factor (NGF) (Merck), 1% glutamax, and 1% penicillin/streptomycin solution. Cultures grown in basal medium (Neurobasal Medium, 2% B-27™ Supplement, 1% glutamax, and 1% penicillin/streptomycin) were taken as control.

Cell growth and viability of HEK293SF cells were compared using two commercial serum-free, chemically defined basal culture media. HyCellTransFx (GE Healthcare, Chicago, IL, USA) and HEK-GM (Xell AG, Bielefeld, Germany) were evaluated in 125 mL shake flasks with 25 mL of initial working volume maintained on an orbital shaker platform (Infor’s HT, Montréal, QC, Canada) at 110 revolutions per minute (rpm) in conditions of 80% humidity, 5% CO₂, and 37 °C. Cultures were seeded at a cell density of 0.25 × 10⁶ cells/mL, and the cells were monitored daily. The addition of feed supplements was also assayed in identical cultures run in parallel, following the manufacturers’ recommendations. Supplementing the HEK-GM basal media consisted of a bolus addition of HEK-FS (Xell AG, Bielefeld, Germany) supplemented with 4 mM GlutaMAX™ (MilliporeSigma, Oakville, Ontario, Canada), starting from day 2 of the culture at 3% (v/v), then increasing to 4 and 5% (v/v), and then 10% (v/v) until the end of the culture. Supplementing the HyCellTransFx medium was carried out with Cell Boost™ 5 (GE Healthcare, Chicago, IL, USA, USA) every second day by adding a bolus at 5% (v/v) supplemented with 4 mM GlutaMAX™ (MilliporeSigma, Oakville, Ontario, Canada) until the end of the culture. Cell counts were performed daily.

The MCF-7 human breast adenocarcinoma cell line was purchased from the Health Protection Agency (European Collection of Cell Cultures EACC, Salisbury, UK). Cells were cultured at 37 °C in an atmosphere containing 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM), which contains a high glucose level (4500 mg/L-glucose), no HEPES, no phenol-red, 5% charcoaled fetal bovine serum (FBS), 1% GlutaMAX and 1% sodium pyruvate (Sigma-Aldrich, St. Louis, Missouri, USA). Stably transfected cells were selected by the supplementation of 200 µg/ml G418 (Thermo Fisher Scientific, Waltham, USA).