Cells were grown to a density of 1–5 × 106 cells per 10 cm dish, washed three times with PBS, and cultured in DMEM low-glucose medium without FBS. After 3 h, culture medium was removed from the dish, and the cells were washed twice in 5% mannitol solution, first with 10 mL and next with 2 mL. Metabolites were extracted from 2.7–3.0 × 106 cells with 800 μL methanol and 550 μL Milli-Q water containing Internal Standard Solution (Human Metabolome Technologies [HMT]) for CE-MS, and with 1300 μL ethanol containing Internal Standard Solution for LC-MS. Extracts for CE-MS were transferred into a microfuge tube and centrifuged at 2,300 × g at 4°C for 5 min. To remove proteins, the extracts were centrifugally filtered through a 5 kDa cutoff filter (Millipore) at 9,100 × g at 4°C for 2 h. Extracts were stored at -80°C until analysis. Before measurement, extracts for CE-MS were centrifugally concentrated and resuspended in 50 μL of Milli-Q water for measurement. Extracts for LC-MS were mixed with 1,000 μL Milli-Q water and sonicated for 5 min while cooling on ice, and the supernatant was collected by centrifugation (4,400 × g, 4°C, 5 min). It was dissolved in 200 μL of 50% aqueous isopropanol solution (v/v) and used for the measurement.
Internal standard solution
Manufactured by Human Metabolome Technologies
Sourced in Japan
The Internal Standard Solution is a prepared solution containing a mixture of compounds that serve as internal standards for metabolomic analysis. This solution is used to accurately quantify the levels of specific metabolites in biological samples by mass spectrometry. The internal standards help to account for variations in sample preparation and injection, allowing for more reliable and reproducible quantification of metabolites.
Automatically generated - may contain errors
Lab products found in correlation
5 kda cutoff filter, by Merck Group (3 mentions)
Agilent ce tofms system, by Agilent Technologies (1 mentions)
Peakstat, by Human Metabolome Technologies (1 mentions)
Samplestat, by Human Metabolome Technologies (1 mentions)
Carcinoscope, by Human Metabolome Technologies (1 mentions)
Ce tofms, by Human Metabolome Technologies (1 mentions)
Milli q, by Merck Group (1 mentions)
8 protocols using internal standard solution
1
Metabolite Extraction for CE-MS and LC-MS Analysis
2
Metabolomic Analysis of 3T3-L1 Cells
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Confluent 3T3-L1 cells in 90-mm tissue culture dishes were incubated for 3 h with 10 mL of DMEM-MG containing 0.1% (v/v) DMSO or 20 μM DIF-1, DIF-1(3M), or CP-DIF-1; the assay was performed in duplicate. The culture media were removed, and the cells were washed with 10 mL per dish of 5% (w/v) mannitol solution and then 2 mL of the same solution. The cells were collected by scraping in methanol (1.3 mL/well) containing 10 μM internal standard solution (Human Metabolome Technologies, Tokyo, Japan) and transferred into eight centrifugation tubes. Ionic metabolites were analyzed by capillary electrophoresis time-of-flight mass spectrometry (Agilent CE-TOFMS system; Agilent Technologies, Waldbronn, Germany) as described previously [23 (link),54 (link),55 (link),56 (link),57 (link)]. Relative quantification data for the identified metabolites were used for hierarchical cluster analysis (HCA) and principal component analysis (PCA) performed with the proprietary software, PeakStat and SampleStat (Human Metabolome Technologies), respectively, to produce a metabolome heat map and a metabolome pathway-map.
3
Metabolic Profiling of Cancer Cells under Hypoxia
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MCF7 and Ishikawa cells stably transfected with COX7RP or empty vector were cultured in normoxic or hypoxic conditions, washed twice with 5% mannitol solution, and the extracts were isolated with methanol containing Internal Standard Solution (Human Metabolome Technologies, Yamagata, Japan). For tracer experiments, cells were cultured in [U-13C]-labeled glutamine for 24 h and the extracts were also isolated. The samples were then passed through a 5-kDa-cutoff filter. The extracted metabolites were concentrated and subjected to CE-TOFMS and triple quadrupole mass spectrometer (QqQ-MS)52 (link). Cellular contents of 2-oxoglutaric acid and succinic acid were also determined using the enzymatic kits for α-ketoglutarate (Sigma-Aldrich) and succinate (Roche, Basel, Switzerland), respectively. NAD(H) levels in mitochondria were estimated using a kit for NAD/NADH (Dojindo, Kumamoto, Japan).
4
Metabolomic Analysis of Intestinal Epithelial Cells
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To measure the metabolites of IECs, isolated IECs from four mice were pooled for each independent measurement. Extracts (>20 mg) were prepared from 106 cells with methanol containing internal standard solution (Human Metabolome Technologies). Cationic compounds were measured in the positive mode of capillary electrophoresis–connected time-of-flight mass spectrometry, and anionic compounds were measured in the positive and negative modes of capillary electrophoresis–tandem mass spectrometry.
5
Metabolite Profiling by CE-MS/MS
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Metabolic methanol extracts spiked with an internal standard solution (Human Metabolome Technologies, Inc., Tsuruoka, Japan) were analyzed using a capillary electrophoresis (CE)-connected ESI-time of flight (TOF)-mass spectroscopy (MS) and CE-MS/MS system (CARCINOSCOPE: Human Metabolome Technologies, Inc.) according to the manufacturer’s instructions39 (link). Concentrations of metabolites were calculated by normalizing the peak area of each metabolite with respect to the area of the internal standard and using standard curves, which were obtained by three-point calibration.
6
Metabolomic Profiling of Neuroblastoma Cells
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NB9 cells and SK‐N‐AS cells were seeded in 10‐cm dishes at a density of 2 × 103 cells/dish and 1 µmol/L 9bw was added 24 h after seeding. At 8 h after addition of 9bw, culture medium was removed from the dishes, and cells were washed in 5% mannitol solution. The cells were then treated with methanol and Internal Standard Solution (Human Metabolome Technologies HMT, Inc) to extract intracellular metabolites. After ultrafiltration to remove proteins, metabolic extracts were analyzed using a capillary electrophoresis‐time of flight mass spectrometry (CE‐TOFMS) system (Agilent Technologies). Peak information, including mass‐to‐charge ratio (m/z), peak area, and migration time (MT), were determined using automatic integration software (MasterHands, Keio University, Tsuruoka, Japan). The peaks were matched with estimated metabolites from the HMT metabolite library and Known‐Unknown peak library based on their m/z values and MTs. The tolerances were set to 10 ppm for m/z and 0.5 min for MT. The concentrations of the metabolites were determined using a standard curve plotted by analyzing standard compounds. For the metabolites without standard compounds, peak areas normalized with internal standards are shown.
7
Liver Metabolite Extraction and Analysis
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Approximately 50 mg of liver was dissected and snap-frozen in liquid nitrogen. The frozen liver was homogenized in 500 lL of cold methanol containing 50 lM internal standard solution (Human Metabolome Technologies, Tsuruoka, Japan). The homogenate was mixed with 500 lL chloroform and 200 lL Milli-Q water and further homogenized. The aqueous layer was collected by centrifugation at 5000 g for 5 min at 4 °C and centrifugally filtered through a Millipore 5-kDa cutoff filter to remove proteins. The filtrate was lyophilized, suspended in 50 lL Milli-Q water and analyzed using CE-TOFMS (Human Metabolome Technologies, Tsuruoka, Japan).
8
Intracellular Metabolite Extraction for CE-MS
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Metabolic extracts of intracellular metabolites were prepared from HCECs cultured in 6-well or 24-well plates with methanol containing Internal Standard Solution (Human Metabolome Technologies, Inc., Tsuruoka, Japan). The culture medium was aspirated from the well, and the cells were washed two times with 5% mannitol solution as follows: 1.5 mL followed by 0.5 mL (6-well plates) or 0.3 mL followed by 0.1 mL (24-well plates). The cells were then treated with 600 lL (6-well plates) or 200 lL (24-well plates) of methanol and left at rest for 30 seconds in order to inactivate enzymes. Next, the cell extract was treated with 410 lL (6-well plates) or 140 lL (24-well plates) Milli-Q (EMD Millipore)-purified water containing internal standards (H3304-1002; Human Metabolome Technologies) and left at rest for another 30 seconds. The obtained extract was then centrifuged at 2300g at 48C for 5 minutes, and all of the upper aqueous layer was centrifugally filtered through a 5-kDa cutoff filter (EMD Millipore) at 9100g and 48C for 120 minutes to remove proteins. The filtrate was then centrifugally concentrated and resuspended in 50 lL Milli-Qpurified water for capillary electrophoresis-mass spectrometry (CE-MS) analysis.
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