Agilent 1100 isocratic hplc pump

CE‐TOFMS was performed using an Agilent CE Capillary Electrophoresis System with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE‐MS adapter kit, and Agilent G1607A CE‐ESI‐MS sprayer kit (Agilent Technologies, Waldbronn, Germany), as described in the previous papers.17, 18, 19, 20 The systems were controlled by the Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies). The metabolites were analyzed by using a fused silica capillary with the electrophoresis buffer (Human Metabolome Technologies) as the electrolyte. The sample was injected at a pressure of 50 mbar for 10 s in cation analysis and 25 s in anion analysis. The spectrometer was scanned from m/z 50 to 1000.

For hydrophilic metabolite analysis, capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) was used for cationic and anionic metabolite analyses. To extract the metabolites, cultured organoids from E7386-0, 30, and 100 nM groups were collected after adding 1.0 ml methanol containing internal standards (25 µM each of methionine sulfone and D-camphor-10-sulfonic acid). The suspension (400 µl) was then mixed with Milli-Q water and chloroform at a volumetric ratio of 5:2:5 and centrifuged at 9,100 × g for 10 min at 20°C. Subsequently, the aqueous layer (400 µl) was centrifugally filtered through a 5 kDa cut-off filter (cat. no. UFC3LCCNB-HMT; Human Metabolome Technologies, Inc.) to remove proteins. The filtrate was centrifugally concentrated and dissolved in 25 µl Milli-Q water containing the reference compounds (200 µM each of 3-aminopyrrolidine and trimesic acid) immediately prior to CE-TOFMS analysis [Agilent 1100 isocratic HPLC pump, Agilent G1603ACE-MS adapter kit, and Agilent G1607A CE-electrospray ionization (ESI)-MS sprayer; Agilent Technologies, Inc.] (13 (link),14 (link)). The quantified data were standardized with the internal standards before being normalized to the DNA concentrations of each sample.

CD3⁺ T cells (around 1.8 × 10⁷ cells per sample) were activated for 36 hours in conditional media (RPMI-1640) containing 2 mM ¹³C₅-glutamine, 1.15 mM ¹³C₆-arginine, or 0.17 mM ¹³C₅-proline, and then used for the extraction of intracellular metabolites as described above. Metabolome measurements were carried out through a facility service at Human Metabolome Technologies Inc. (Tsuruoka, Japan). CE-TOFMS measurement was carried out using an Agilent CE Capillary Electrophoresis System equipped with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). The systems were controlled by Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies, Waldbronn, Germany). The metabolites were analyzed by using a fused silica capillary [50 μm internal diameter (i.d.)× 80 cm total length], with commercial electrophoresis buffer (solution ID: H3301-1001 for cation analysis and H3302-1021 for anion analysis, Human Metabolome Technologies) as the electrolyte. The sample was injected at a pressure of 50 mbar for 10 s (approximately 10 nl) in the cation analysis and 25 s (approximately 25 nl) in the anion analysis. The spectrometer was scanned from m/z 50 to 1000.