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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.

Endogenous FA metabolites were measured using a triple quadrupole mass spectrometer LCMS-8060 (Shimadzu) as previously described (25 (link), 55 (link)). Briefly, a reverse-phase column (Kinetex C8, 2.1 × 150 mm, 2.6 μm, Phenomenex) was used for chromatographic separation with a binary mobile phase comprising 0.1% formic acid/water (mobile phase A) and acetonitrile (mobile phase B). The gradient of the mobile phase (%A/%B) was programmed as follows: 0 min (90/10), 5 min (75/25), 10 min (65/35), 20 min (25/75), 20.1–28 min (5/95), 28.1–30 min (90/10). The flow rate was 0.4 ml/min, and the column temperature was 40 °C. The selected reaction-monitoring transitions are listed in Supplementary Table 1. Raw data were analyzed by using LabSolutions Insight (Shimadzu). The signals were compared with the standard curves for quantification. Although each sample containing six (respectively 8) DRGs was analyzed using LC-MS, the graphs show the values of metabolites calculated per one DRG.

SCFAs and other metabolites were extracted and measured as previously described (60 (link)). In brief, 5 mg of feces or colonic contents were lyophilized. Dried samples were added to 5 μl Milli-Q water containing internal standards (2.2 mM [1,2-13C2]acetate, 2.2 mM [2H7]butyrate and 2.2 mM crotonate), 50 μl HCl and 200 μl diethyl ether. After centrifugation, 80 μl of the organic layer was transferred to a glass vial and then 16 μl of N-tert-butyldimethylsilyl-N-trifluoroacetamide (Sigma-Aldrich) was added to derivatize the samples. The vials were incubated at 80°C or 20 min and allowed to stand for 48h before injection. The analysis was performed using a gas chromatography-tandem mass spectrometry (GCMS) platform on a Shimadzu GCMS-TQ8040 triple quadrupole mass spectrometer (Shimadzu) with a capillary column (BPX5) (SGE Analytical Science). The program of GCMS analysis is described in a previously published paper (60 (link)). The GCMS data were processed, and concentrations were calculated by LabSolutions Insight (Shimadzu).