We performed metabolomic analysis using C‐Scope (Human Metabolome Technologies), according to the recommended protocol.23 (link) Rh30 cells administered with DMSO or MCDi for 24 h were used as samples. In brief, after washing twice with 5% mannitol solution, 800 µl methanol, and 550 µl of 8 µM internal standard were added to the cells. Next, the cells were centrifuged at 4℃, 2300 × g for 5 min. The supernatants were then collected by centrifugation through a 5 kDa‐cutoff filter at 4℃, 8100 × g for 3 h. The concentrations of all charged compounds were measured by capillary electrophoresis time‐of‐flight mass spectrometry (CE‐TOFMS) and capillary electrophoresis tandem mass spectrometry (CE‐QqQMS; CE‐MS/MS) according to a previously described method.24 (link)
C scope
Manufactured by Human Metabolome Technologies
Sourced in Japan
The C-Scope is a high-performance liquid chromatography (HPLC) system designed for the analysis of metabolites. It features a compact and modular design, allowing for efficient separation and detection of a wide range of metabolic compounds.
Automatically generated - may contain errors
8 protocols using c scope
1
Metabolomic Analysis of Rh30 Cells
2
Metabolite Analysis by CE-MS/MS
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Metabolite analysis was performed using C-Scope (Human Metabolome Technologies, Yamagata, Japan), according to the recommended protocol [41 (link)]. Briefly, the cells were washed twice with 5% mannitol solution and treated with 0.8 ml of methanol and 0.55 ml of 8 μM internal standard (IS). After centrifugation at 2,300 g, 4°C for 5 min, the supernatants were collected for centrifugal filtration through a 5-kDa-cutoff filter at 9,100 g, 4°C for 3 h. The extracted metabolites were stored at −80°C until analysis. Concentrations of all charged compounds were measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and capillary electrophoresis tandem mass spectrometry (CE-QqQMS; CE-MS/MS) as described previously [41 (link), 42 (link)].
3
Intracellular Metabolite Analysis by GC/MS and CE-MS
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FLS intracellular metabolites (0.2–1.0 × 106 cells) were analyzed by gas chromatography-mass spectrometry (GC/MS) and capillary electrophoresis-mass spectrometry (CE-MS; C-SCOPE, Human Metabolome Technologies Inc., Tsuruoka, Japan). GC/MS analysis was performed using a GC/MSQP2010 Ultra (Shimadzu Co., Kyoto, Japan) with a fused silica capillary column (CP-SIL 8 CB low bleed/MS; 30 m × 0.25 mm inner diameter, 0.25 μm film thickness; Agilent Technologies, Waldbronn, Germany), as described previously [34 (link), 35 (link)]. 2-Isopropylmalic acid solution (Sigma-Aldrich) was added as an internal standard. The C-SCOPE analysis was performed as described previously [36 (link)]. 5-Hydroxy-N-methyl-tryptamine (Human Metabolome Technologies Inc.) was added as an internal standard.
4
Comprehensive Metabolome Analysis of Tissues
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Metabolome analysis was performed using a facility service (C-SCOPE) at Human Metabolome Technologies, Inc. (Yamagata, Japan). Briefly, the tissues were homogenized in a 50% acetonitrile aqueous solution. The samples were then centrifuged at 2,300 × g at 4˚C for 5 min and the upper aqueous layer was centrifugally filtered through a 5 kDa cutoff filter at 9,100 × g for 120 min at 4˚C. The filtrates were resuspended in 50 μl of Milli-Q water and the metabolome analysis was performed using CE-TOFMS and CE-QqQMS. The data were analyzed by MasterHands ver.2.17.1.11 (Keio University, Tsuruoka, Japan) and MassHunter Quantitative Analysis B.06.00 (Agilent Technologies, Santa Clara, CA), and the peak area of each metabolite was calculated. The peak area was then normalized to an internal standard, and the metabolite concentrations were calculated using standard curves. Overall, 116 compounds were measured and 30 metabolic parameters were calculated.
5
Metabolome Analysis of Adherent Cells
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Metabolome analysis was carried out through a facility service(C-SCOPE) at Human Metabolome Technologies, Inc.(HMT, Inc., Japan) as previously described (n=3 in each group) [25 (link), 26 (link)]. A total of 116 metabolites were analyzed. When collecting samples, adherent culture cells were removed using a cell scraper in an effort to exclude the possible influence of the dissociation solution. They were subsequently treated with protocols identical to those employed with spheroids. Washes were performed using 5% mannitol.
6
Metabolomic Analysis of CD34+ Cells
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CD34+ cells were isolated from the CB or BM using the Indirect CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer's instructions. Metabolite extraction was performed according to the manual of Human Metabolome Technologies. Metabolites were analyzed using CE‐TOFMS systems (C‐Scope, Human Metabolome Technologies). More details about the experiments are described in our previous study.29
7
Metabolomics of Alport Syndrome Mouse Model
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Metabolome analysis was performed using C-Scope [Human Metabolome Technologies (HMT), Yamagata, Japan] according to the recommended protocol. In brief, kidney tissue samples were collected from WT and metformin- or losartan-treated C57BL/6 Col4a5 G5X Alport syndrome mice, and quickly frozen in liquid nitrogen. Frozen kidney tissues were treated with 50% (v/v) acetonitrile water solution. Then, 10 µM HMT internal standard solution was added and kidney tissues were freeze crushed. After tissue crushing, samples were centrifuge at 2300g, 4 °C for 5 min. Supernatants were collected and filtered using Ultrafree MC PLHCC 5 kDa (HMT, Yamagata, Japan). Filtered sample solutions were dried and resuspended with deionized water. Metabolome analysis was performed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and capillary electrophoresis tandem mass spectrometry (CE-QqQMS; CE-MS/MS).
8
Metabolomic Analysis of DMSO and MCDi Treated Rh30 Cells
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We performed metabolomic analysis using C-Scope (Human Metabolome Technologies, Yamagata, Japan), according to the recommended protocol [47] . Rh30 cells administered with DMSO or MCDi (10 µM) for 24 h were used as samples. In brief, after washing twice with 5% mannitol solution, 800 µl methanol and 550 µl of 8 µM internal standard were added to the cells. Next, the cells were centrifuged at 4°C, 2,300 x g for 5 min. The supernatants were then collected by centrifugation through a 5 kDa-cutoff lter at 4°C, 8,100 x g for 3 h. The concentrations of all charged compounds were measured by capillary electrophoresis time-of-ight mass spectrometry (CE-TOFMS) and capillary electrophoresis tandem mass spectrometry (CE-QqQMS; CE-MS/MS) according to a previously described method [48] .
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