Lc ms ms method package for cell culture profiling

We analyzed changes in the concentration of amino acids and glucose in plasma-treated cell culture medium using an LC-MS/MS system (Shimadzu Corp., Nexera X2/LCMS-8050) as described previously with slight modifications^{45 (link)}. The LC/MS/MS Method Package for Cell Culture Profiling (Shimadzu Corp.) provides optimized analytical conditions for up to 95 compounds, either as basal cell culture media components or as secreted metabolites, using LC-MS/MS data. In addition, changes in the mass spectrum of an amino acid in plasma-treated amino acid solution were analyzed with a liquid chromatography mass spectrometer (Shimazu Corp., LCMS-2020). The operating conditions of the electrospray ionization mass spectrometer (ESI-MS) were as follows: (1) electrospray potential = 4.50 kV, (2) capillary voltage = 0 V, (3) capillary temperature = 250 °C, (4) detector voltage = −1.30 kV, (5) skimmer voltage = 0 V, and (6) m/z scan range = 10–300, where m and z represent mass and charge number, respectively. The mass resolution of the LCMS-2020 as defined by m/Δm_50% is 2 m, where Δm_50% is the peak width at 50% of the maximum peak intensity⁴⁶ .

Four testicular fragments from a P7 HV-Tg rat were placed on a 1.5% agarose gel block without a PC device as described above. The first day of culture was designated as day 0. 30 µL of medium was collected from each well on days 3, 7, 14, 17, 21, 24 and 28. On days 7, 14, 21 and 28, the culture medium was fully changed after collecting the sample. The LC/MS/MS method package for cell culture profiling (version 2; Shimadzu, Kyoto, Japan) was used for the metabolomics analysis. Briefly, 40 µL of Milli-Q water, 10 µL of 0.5 mM isopropylmalic acid (333115, Sigma) as an internal standard, and 100 µL of acetonitrile (018-19853, Fujifilm, Tokyo) were added to 10 µL of standard or sample medium and vortexed. 50 μL of the supernatant of the sample after centrifugation at 15,000 rpm for 15 min at room temperature was collected and diluted with 450 μL of Milli-Q water, and 1 μL was introduced into an LC (Nexera X3; Shimadzu) with a Discovery HS F5-3 column (567503-U, Merck; 2.1 mm I.D. × 150 mm, 3 μm) connected to a tandem quadrupole mass spectrometer (LCMS-8060NX; Shimadzu). The chromatographic parameters were set according to the instruction manual (225-41626A; Shimadzu). LabSolutions Insight (Version 3.7 SP3; Shimadzu) was used to analyze the chromatographic data.