N acetylaspartic acid

Differentiated iBACs (day 7–8 of differentiation) were harvested in homogenization buffer (PBS pH 8.5, 1 mM DTT, 10% glycerol) and sonicated 3× for 5 s. Cell homogenates were centrifuged for 10 min/10000 × g/4 °C and supernatants were collected. An equal amount of 2× reaction buffer (dH₂O pH 8.5, TrisHCl 100 mM, NaCl 100 mM, CaCl₂ 5 mM, DTT 0.2 mM, IGEPAL CA-630 (1%, Sigma) 0.5%, N-acetylaspartic acid (Sigma) 40 mM) was added to the samples and a defined volume per sample was collected to provide the same background matrix for the aspartate standard curve. The background matrix was heated at 95 °C for 10 min and subsequently centrifuged for 3 min/16000 × g/4 °C. The background supernatant was collected and all samples and background sample were incubated for 18 h/38 °C/350 rpm in a thermomixer. After incubation, all samples were inactivated at 95 °C for 10 min and subsequently centrifuged for 3 min/16000 × g/4 °C. Supernatants were used for analyzing released aspartate content using the aspartate assay kit (BioVision).

Creatine (Sigma Aldrich, C0780), creatinine (Sigma Aldrich, 1052060050), taurine (Sigma Aldrich, T8691), guanidine acetate (Sigma Aldrich, 50,920), carnitine (Sigma Aldrich, C9500), 4-quinolinecarboxylic acid (Sigma Aldrich, 174823), quinoline-2,4-dicarboxilic acid (Sigma Aldrich, 8151030025), DL-2-aminoadipic acid (Sigma Aldrich, A0637), N1-acetylspermidine (Cayman Chemical 9001535), N-acetyl aspartic acid (Sigma Aldrich, 00920) were used.