L glutamate

HEK293T cells were cultured in DMEM medium (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone), 2 mM L-glutamate (Lonza), 100 μg/ml penicillin-streptomycin (Lonza), 1 mM sodium pyruvate (Lonza), and NEAA (Gibco, Waltham, MA, USA).
All primary cells were maintained in RPMI 1640 (Lonza) medium supplemented with 7.5% heat-activated fetal bovine serum (FBS, Hyclone), 2 mM L-glutamate (Lonza), 100 μg/ml penicillin-streptomycin (Lonza), and 50 μM β-Mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA). All cells were cultured at 37°C in a humidified atmosphere of 5% CO₂.

Five human mesenchymal stem cell (hMSCs) populations isolated from the iliac crest of normal donors were obtained from Lonza (Australia) and expanded as a monolayer culture. These cells were obtained through informed consent (see manufacturer’s supporting documentation (USWV-10276)) and have been used previously in numerous studies [38 (link)–40 (link)]. Cultures were maintained in human mesenchymal stem cell growth media (hMSCGM) containing basal medium (hMSCBM) supplemented with 10% human mesenchymal stem cell growth supplement, 100U/mL gentamycin/ampicillin and 10% L-glutamate (Lonza, Australia). Cells were grown in a 5% CO₂ humidified atmosphere at 37°C and plated at 3000 cells/cm² in 100mm culture dishes (Corning, Australia) in maintenance media. For immunocytochemistry (ICC) experiments cells were grown in CC2 coated, 8 chamber glass slides (Labtek, Australia). For Q-PCR experiments cells were plated at 3000 cells/cm² in 6 well plates (3x10⁴ per well; Corning) and RNA harvested after three days.