Glutamic acid

The content of free AAs was determined by the ninhydrin dyeing method as described by Yin et al. (2009) (link) with minor modifications using Glutamic acid as standard. Ninhydrin solution (40 g L^–1) of 0.5 mL was added to 1.0 mL of tea extract in a test tube with cap. Then 0.5 mL of pH 8.0 buffer (95% v/v 0.067 mol L^–1 Na₂HPO₄⋅12H₂O solution and 5% v/v 0.067 mol L^–1 KH₂PO₄ solution) was added to the mixture. Test tubes with caps were then placed in a water bath at 100°C for 15 min. After cooling to room temperature, the solution was transferred to a volumetric flask and diluted to 25 mL with deionized water. Absorbance of the diluted solution at 540 nm was determined using a 1 cm photometer disposable cuvette and a spectrophotometer. Glutamic acid, ninhydrin, Na₂HPO₄⋅12H₂O, KH₂PO₄ were equal or above to ACS grade, purchased from Thermo Fisher Scientific.

DPBS was purchased from HyClone (Logan, UT). Aspartic acid, CsOH, Cs₂SO₄, CsCl and D-mannitol were from Sigma. Tetraethylammonium chloride (TEA-Cl), 1.0 M CaCl₂ and 1.0 M MgCl₂ were from Fluka Honeywell (Muskegon, MI). Glutamic acid, HEDTA, HEPES sodium, tetramethylammonium chloride (TMA-Cl) and NaCl were from Thermo Fisher. HEPES acid and sodium aspartate were from Alfa Aesar (Ward Hill, MA). KCl was from Fisher Scientific. Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) was from Promega (Madison, WI).

Metabolic profiling of O9-1 cells was performed using a Seahorse XFe Analyzer (Agilent). O9-1 cells were seeded in preconditioned medium into Seahorse XFe 96-well plates at a density of 20 000 cells per well and incubated overnight. Before the measurement, the cells were washed twice and incubated in fresh XF DMEM pH7.4 (Agilent) supplemented with 10 mmol·L⁻¹ glucose (Carl Roth), 1 mmol·L⁻¹ sodium pyruvate (Gibco) and 2 mmol·L⁻¹ glutamic acid (Gibco) for 45 min at 37 °C without CO₂. The cartridge was hydrated with XF Calibrant solution (Agilent) for 16 h, loaded with 75 μmol·L⁻¹ oligomycin and 25 μmol·L⁻¹ rotenone + antimycin A before measurement according to Agilent’s ATP-Rate assay protocol. After the measurement, the cells were washed with PBS, treated with 70% ethanol for 30 min and stained with crystal violet. The obtained cell density values were used for data normalization. Total ATP generation as well as mitochondrial and glycolytic contributions were determined using the Wave Desktop Application (Agilent) for the conducted assay.

Primary hippocampal neurons were cultured from Wistar rat E18 embryos. Hippocampi were removed from embryonic brains, placed in HBSS (Gibco, Life Technologies, NY) and incubated with 0.25% trypsin (Life Technologies) and 0.1% DNase (Roche Applied Science, Penzberg, Germany) for 15 min at 37°C. Hippocampi were then mechanically dissociated using gentle pipetting. Neuronal cells were either plated at 20,000–50,000 cell/cm² or electroporated using the Nuclefector Amaxa system (Lonza, Basal, Switzerland, Rat Neuron Nuclefector Kit, CN # VPG-1003) according to manufacturer’s instructions then plated at 50,000 cell/cm². Cells were plated in MEM + Glutamax (Gibco) containing 3% glucose and 10% serum and then switched to standard culturing media (Neurobasal media (Gibco) with 1% Pen/Strep/Glut (Life Technologies), Glutamic acid (Life Technologies) and 2% B27 (Gibco) or 2% SMI (Stemcell Technologies, Vancouver, Canada)) 2–4 hr following plating. Cell were plated in LabTek II Chambered Coverglass chambers (Nunc, Rochester, New York), 96-well glass bottomed plates (In Vitro Scientific, Sunnyvale, CA) or 96-well plastic bottomed plates (Costar, Corning, New York) coated with poly-l-lysine (0.1 mg/mL, MW 30,000–70,000 Sigma Aldrich) or poly-l-lysine and laminin (2 μg/mL, Mouse Protein, Natural, Life Technologies).

SCG explants were dissected from E16 mouse embryos and seeded in FluoroDish dishes (World Precision Instruments, Sarasota, FL, USA) coated with poly-L-ornithine (0.5 mg/ml) and laminin (5 μg/ml). Explants were cultivated in neurobasal medium (Life Technologies, Waltham, MA, USA) containing B27 serum (2%, Life Technologies, Waltham, MA, USA), Glutamine (2 mM, Life Technologies, Waltham, MA, USA), Glutamic acid (25 µM, Life Technologies, Waltham, MA, USA), Penicillin-Streptomycin (200 U/ml, Life Technologies, Waltham, MA, USA) and Ngf (10 ng/ml, Life technologies, MNAC-25, Waltham, MA, USA). After 24 hr in culture, explants were electroporated with a Rab5-GFP plasmid (Roberts et al., 1999 (link)) (500 ng/µl) in PBS 1X using a BTX Electro Square Porator ECM 830 (2 times 3 pulses of 200 V/cm, separated by intervals of 15 ms). The movements of Rab5-GFP⁺ vesicles were recorded 24h after electroporation on a confocal microscope (Leica SP5-SMD, Germany) at 37°C in a humidified atmosphere containing 5% CO₂ for 2 min for each acquisition.