Pump system

The quantification of amino acid concentrations in spinal cord tissue homogenate was performed by HPLC. Briefly, spinal cord homogenate was filtered through 200 μm filters and diluted at 1:10 ratio. Two-hundred and forty microliters of diluted sample were added to HPLC tubes. All mobile phases (polar and apolar) for elution were filtrated and degasified at least for 45 min. The mobile polar phase was composed by disodium phosphate, propionic acid and acetonitrile; and the mobile apolar phase by acetonitrile and methanol. A precolumn derivatization method using ortho-phthalaldehyde 1:5 [(OPA) with methanol ≥99.9%, potassium borate 1 M pH 9.5, and 2-β-mercaptoethanol ≥99.0% (Sigma-Aldrich, USA)] to detect amino acids in a Gilson UV/vis_155 detector (338 nm) was used. Moreover, a Gilson pump system (Gilson) was used with a 40 °C Hi-Chrom C18 (model HI-5C18-250A) 5 μm particles column (Hi-Chrom) (Thermo Fisher Scientific, USA). All data was analyzed using the Gilson Uniprot Software version 5.11. Standard solutions of each amino acid were prepared in MilliQ water (Millipore, USA): l-aspartate, l-glutamate, l-asparagine, Histidine, l-serine, l-glutamine, l-arginine, Glycine, l-alanine, l-lysine, l-isoleucine, l-phenylalanine, l-methionine, l-tryptophan, l-cistine, and l-leucine (all from Sigma-Aldrich, USA).

After infection, supernatants were removed, centrifuged, and transferred to high-performance liquid chromatography (HPLC) tubes. Glucose and lactate levels were determined using a Gilson pump system (Gilson) with a 54°C HyperREZ XP Carbohydrate H⁺ 8-μm (Thermo Fisher Scientific) column and a refractive index detector (IOTA 2; Reagents). The mobile phase consisting of 0.0025 M H₂SO₄ was filtered and degasified for at least 45 min before use. Standard solutions were prepared in MilliQ water (Millipore). All data were analyzed using the Gilson Uniprot software, version 5.11.