Ultrafreemc plhcc

The cell suspension was transferred to a tube and centrifuged to pellet the cells. Culture medium was aspirated from the tube, and the cells were washed with 10 ml of 5% mannitol solution. The cells were then treated with 800 µL of methanol and vortexed for 30 sec to suppress enzyme activity. Next, 550 µL of Milli-Q water containing internal standards (H3304-1002, Human Metabolome Technologies, Inc. (HMT), Tsuruoka, Yamagata, Japan) was added to the cell extract, which was vortexed for another 30 s. The extract was then centrifuged at 2300 × g, 4 °C for 5 min, after which 700 µL of the supernatant was centrifugally filtered through a Millipore 5-kDa cutoff filter (UltrafreeMC-PLHCC, HMT) at 9100 × g, 4 °C for 120 min to remove macromolecules. Subsequently, the filtrate was evaporated to dryness under vacuum and reconstituted in 50 µL of Milli-Q water for metabolome analysis at HMT. Metabolome analysis was conducted according to HMT’s ω Scan package, using capillary electrophoresis Fourier transform mass spectrometry (CE-FTMS) based on the methods described previously [23 (link)].

For metabolome analysis, mice were fasted for 12 h and sciatic nerves were extracted soon after euthanasia and kept at – 80°C. These samples were analyzed by Human Metabolome Technologies, Inc. (Tokyo, Japan) using a capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) system. In brief, frozen sciatic nerve tissue was placed in a homogenization tube, along with zirconia beads. Next, 1200–1500 μl of 50% acetonitrile/Milli-Q water containing internal standards (H3304-1002, Human Metabolome Technologies, Inc. (HMT), Yamagata, Japan) was added to the tube, after which the tissue was completely homogenized at 3500 rpm, 4°C for 540 s using a bead shaker (Shake Master NEO, Bio Medical Science, Tokyo, Japan). The homogenate was then centrifuged at 2300 × g for 5 min at 4°C. Subsequently, 400 μl of the upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter (UltrafreeMC-PLHCC, HMT) at 9,100 × g, 4°C for 180 min to remove macromolecules. The filtrate was evaporated to dryness under vacuum and reconstituted in 50 μl of Milli-Q water for metabolome analysis at HMT. Metabolome analysis was conducted according to HMT’s Basic Scan package using CE-TOFMS, based on the methods.⁴⁹ (link)^,50 (link)

The culture medium was aspirated from the dish, and the cells were washed twice with a 5% mannitol solution (10 mL and 2 mL for the first and second wash, respectively). The cells were then treated with 800 µL of methanol and incubated at room temperature for 30 sec to suppress enzyme activity. Next, 550 µL of Milli-Q water containing internal standards (H3304-1002, Human Metabolome Technologies, Inc. (HMT), Tsuruoka, Yamagata, Japan) was added to the cell extract, followed by further incubation at room temperature for 30 s. The cell extract was then centrifuged at 2300× g at 4 °C for 5 min, after which 700 µL of the supernatant was centrifugally filtered through a Millipore 5-kDa cutoff filter (UltrafreeMC-PLHCC, HMT) at 9100× g at 4 °C for 120 min to remove macromolecules. Subsequently, the filtrate was evaporated to dryness under a vacuum and reconstituted in 50 µL of Milli-Q water for metabolome analysis at HMT.

For extracting ionic metabolites, approximately 50 mg of cecal contents was dissolved in MilliQ water containing internal standards (H3304-1002, Human Metabolome Technologies (HMT), Tsuruoka, Yamagata, Japan) at a ratio of 1:9 (w/v). After centrifugation, the supernatant was centrifugally filtered through a Millipore 5000-Da cutoff filter (UltrafreeMC-PLHCC, HMT) to remove macromolecules (9100×g, 4 °C, 60 min) for subsequent analysis with capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS).

Extraction and measurement of metabolites excluding glycogen assay were performed in the faculty of Human Metabolome Technologies (HMT, Tsuruoka, Yamagata, Japan) Inc. Approximately 20–40 mg of frozen tissue was placed in a homogenization tube together with zirconia beads (5 mmφ and 3 mmφ). Next, 1,500 µL of 50% acetonitrile/Milli-Q water containing internal standards (H3304-1002, HMT) was added to the tube, after which the tissue was completely homogenized at 1,500 rpm for 60 s at 4°C using a beads shaker (Shake Master NEO, Bio Medical Science, Tokyo, Japan). The homogenate was then centrifuged at 2,300 g for 5 min at 4°C. Subsequently, 800 µL of the upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter (UltrafreeMC-PLHCC, HMT) at 9,100 g for 180 min at 4°C to remove macromolecules. The filtrate was evaporated to dryness under vacuum and reconstituted in 50 µL of Milli-Q water for metabolome analysis at HMT.