Alanine

All reagents were purchased from Sigma Aldrich (St. Louis, MI, USA) if not stated otherwise. A standard mixture of 17 amino acids, namely histidine, serine, arginine, glycine, aspartate, glutamate, alanine, threonine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, and phenylalanine, was acquired from Waters (Milford, MA, USA). Asparagine, glutamine, taurine, histamine, ethanolamine, methylamine, citrulline, ethylamine, ornithine, putrescine, serotonin, cadaverine, tyramine, tryptamine, and 2-phenylethyl-amine standards were obtained from Sigma-Aldrich (St. Louis, MI, USA), and tryptophan was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA).

Quantification of amino acids in cell and media samples was performed using the Waters Acquity UPLC H-Class system (Waters, Milford, MA, USA). This method combines pre-column sample derivatisation using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), ultra performance liquid chromatography for chromatographic separation and subsequent UV detection.
The 18 individual amino acid standards including histidine (His), serine (Ser), arginine (Arg), glycine (Gly), aspartate (Asp), glutamate (Glu), threonine (The), alanine (Ala), proline (Pro), cystine (Cys), lysine (Lys), tyrosine (Tyr), methionine (Met), valine (Val), isoleucine (Ile), leucine (Leu) and phenylalanine (Phe) were purchased from Waters. Glutamine (Gln) standard was purchased from Sigma-Aldrich (Dorset, UK). The standards mixtures were made up into a stock solution containing 0.25 mM of each amino acid except for cystine (0.125 mM).
In the optimized culture conditions, asparagine (Asn) had also been added to the amino acid quantification method used here. A stock solution containing 0.25 mM Asn was used.