Media samples at day 16 (2-days post-treatment) for cells treated with 0.001–0.1 μM bortezomib, and 0.01–1 μM tamoxifen as well as untreated control were shipped for analysis to Human Metabolome Technologies America, Inc. (HMT; Boston, MA, USA). For sample preparation, 40 μL of samples were mixed with 10 μL of Milli-Q water containing internal standards (1,000 μM). The mixture was then filtered through a 5 kDa cut-off filter (ULTRAFREE-MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan) to remove macromolecules. Metabolome analysis was performed in samples of culture medium using Capillary Electrophoresis Time-of-Flight Mass Spectrometry (CE-TOFMS) in two modes for cationic and anionic metabolites. On the basis of HMT's standard library, 65 metabolites (47 metabolites in Cation mode and 18 metabolites in Anion mode) were detected (dataset provided in Supplementary Materials ).
Ultrafree mc plhcc
Manufactured by Human Metabolome Technologies
Sourced in Japan
The ULTRAFREE-MC-PLHCC is a centrifugal filtration device designed for the separation and concentration of samples. It features a low-binding hydrophilic polypropylene membrane and is suitable for use with aqueous solutions, including biological fluids.
Automatically generated - may contain errors
Lab products found in correlation
Lc ms grade, by Fujifilm (2 mentions)
Milli q water, by Human Metabolome Technologies (1 mentions)
H3304 1002, by Human Metabolome Technologies (1 mentions)
Hybrid spe phospholipid 55261 u, by Merck Group (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Chloroform, by Nacalai Tesque (1 mentions)
13 protocols using ultrafree mc plhcc
1
Metabolomic Profiling of Bortezomib and Tamoxifen Effects
2
Targeted Metabolic Profiling of Fibroblasts
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Targeted quantitative analysis of 116 metabolites belonging to glycolysis, pentose phosphate pathway, tricarboxylic acid (TCA) cycle, urea cycle and polyamine, creatine, purine, glutathione, nicotinamide, choline and amino acid metabolisms was performed on fibroblasts using capillary electrophoresis mass spectrometry (CE-TOFMS and CE-QqQMS) in the cation and anion analysis modes. The analysis was performed by Human Metabolome Technologies (HMT).
Briefly, 4.2–4.5 × 106 cells were collected, rinsed with mannitol (5%) and sent to HMT laboratories. The samples were mixed with 800 μl of methanol. Then, 550 μl of Milli-Q water containing internal standards (8 μm ) was added, mixed thoroughly and centrifuged (2300g, 4°C, 5 min). The water layer (350 μl) was filtrated through 5-kDa cut-off filter (ULTRAFREE-MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan) to remove macromolecules. The filtrate was centrifugally concentrated and resuspended in 50 μl of ultrapure water immediately before the measurement. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were performed by statistical analysis software (developed at HMT).
The pathway analysis was performed by the MetaboAnalyst tool (http://www.metaboanalyst.ca ) (73 (link)).
Briefly, 4.2–4.5 × 106 cells were collected, rinsed with mannitol (5%) and sent to HMT laboratories. The samples were mixed with 800 μl of methanol. Then, 550 μl of Milli-Q water containing internal standards (8 μ
The pathway analysis was performed by the MetaboAnalyst tool (
3
Metabolite Analysis by CE-TOFMS
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The supernatants of the KUHIMM culture were filtered through a 5-kDa cut-off filter (ULTRAFREE-MC-PLHCC; Human Metabolome Technologies, Yamagata, Japan), and the filtrates were concentrated by centrifugation and resuspended in 50 μL of ultrapure water immediately before measurement. All metabolome measurements were performed using Capillary Electrophoresis Time-of-Flight Mass Spectrometry (CE-TOFMS) at a facility service at Human Metabolome Technologies Inc55 (link).
4
Metabolomic Analysis of Mouse Salivary Gland
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Metabolome analyses were performed for 3 samples of mouse submandibular salivary gland tissue (wild type SAMP1/kl, 4-week-old SAMP1/kl -/-, and 8-week-old SAMP1/kl -/-) using CE-TOFMS in two modes for cationic and anionic metabolites. The samples were mixed with 50% acetonitrile in water (v/v) containing internal standards (20 μM for cation and 5 μM for anion measurement) and homogenized by a homogenizer (1500 rpm, 120 s × 4 times). The supernatant (400 μL × 2) was then filtered through a 5-kDa cut-off filter (ULTRAFREE-MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan) to remove macromolecules.
5
Metabolite Extraction and Purification for Mass Spectrometry
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Culture medium in Φ10 cm dishes was aspirated and cells were washed twice with 5% mannitol solution (10 mL followed by 2 mL). Cells were treated with 800 μL of methanol and left for 30 seconds to inactivate enzymes. The cell extract was then treated with 550 μL of Milli-Q water containing internal standards (H3304-1002; Human Metabolome Technologies, Tsuruoka, Japan) and left for another 30 seconds. The extract was centrifuged (2300 × g at 4°C for 5 minutes) and then 800 μL of the upper aqueous layer was centrifugally filtered (9100 × g at 4°C for 120 minutes) through a Millipore 5-kDa cutoff filter (UltrafreeMC-PLHCC, Human Metabolome Technologies) to remove macromolecules. The filtrate was centrifugally concentrated and re-suspended in 50 μL of ultrapure water. The obtained samples were sent to Human Metabolome Technologies for analysis by capillary electrophoresis time-of-flight mass spectrometry.
6
Comprehensive Metabolomic Analysis of Kidney Tissue
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Metabolome analyses were performed for 3 samples of mouse kidney tissue (SAMP1/kl wild type, SAMP1/kl-/-, and HL156A-treated klotho-/-) using Capillary Electrophoresis Time-of-Flight Mass Spectrometry (CE-TOFMS) in two modes for cationic and anionic metabolites. The samples were mixed with 50% acetonitrile in water (v/v) containing internal standards (20 μM for cation and 5 μM for anion measurement) and homogenized by a homogenizer (1,500 rpm, 120 sec × 4 times). The supernatant (400 μL × 2) was then filtrated through a 5-kDa cut-off filter (ULTRAFREE-MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan) to remove macromolecules.
7
Metabolomic Profiling of PASMCs
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Metabolomic analyses were done according to the method of Soga et al. by using capillary electrophoresis/mass spectrometry (CE/MS), as described previously [13 (link)]. Briefly, 1,000,000 PASMCs in a 6-cm dish were treated with si-RNA as described above, washed twice with ice cold 5% mannitol, and let stand for 10 min at room temperature in methanol containing L-methionine sulfone (25 μM, Alfa Aesar A17027), MES (25 μM, Dojindo 341–01622), and CSA (25 μM, Wako 037–01032). Then the cells were harvested with a cell scraper and 400 μL of supernatant was collected after vortexing for 30 seconds. After addition of CHCl3 (400 μl) and distilled water (200 μl) with thorough mixing, centrifugation was performed at 10,000 g for 3 minutes at 4°C. Next, the aqueous layer (400 μl) was transferred to an ultrafiltration tube (UltrafreeMC-PLHCC, Human Metabolome Technologies, UFC3LCCNB-HMT), followed by centrifugation at 9,100 g for 3 hours at 4°C. Finally, 320 μL of the filtrate was sent to the Institute for Advanced Biosciences at Keio University for further analyses.
8
Muscle Metabolite Extraction and Preparation
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Approximately 30 mg of muscle tissue sample was mixed with 750 µL of 50% acetonitrile in water (v/v) containing methionine sulfone and camphor-10-sulfonic acid as an internal standard (20 μM) and was homogenized at 3500 rpm (60 s × 5). The same quantity of 50% acetonitrile in water (v/v) was then added to the homogenized sample. Subsequently, 400 µL of supernatant was filtered using a 5-kDa cut-off filter (Ultrafree-MC-PLHCC; Human Metabolome Technologies, Yamagata, Japan) to remove macromolecules. The filtrate was concentrated via centrifugation and resuspended in 50 µL of ultrapure water immediately before analysis.
9
Metabolite Profiling by CE-TOFMS and LC-TOFMS
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For CE-TOFMS analysis, 50 μl of the sample was mixed with 450 μl of methanol containing 10 μM internal standards. Then, 500 μl chloroform and 200 μl Milli-Q water were added, and the mixture was centrifuged at 2300 g, 4 °C for 5 min. The water layer (400 μL) was filtrated through a 5 kDa cut-off filter (ULTRAFREE-MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan) to remove macromolecules. The filtrate was then concentrated using centrifugation and resuspended in 50 μL of ultrapure water immediately before the measurement.
For LC-TOFMS analysis, each 100 μL sample was mixed with 300 μL of 1% formic acid in acetonitrile (v/v) containing 3 μM internal standards and centrifuged at 2,300 g, 4 °C for 5 min. Then phospholipids was removed by filtering the supernatant using a column (Hybrid SPE phospholipid 55261-U, Supelco, Bellefonte, PA, USA). The filtrate was desiccated and resuspended in 100 μL of 50% isopropanol in Milli-Q water (v/v) immediately before the measurement.
For LC-TOFMS analysis, each 100 μL sample was mixed with 300 μL of 1% formic acid in acetonitrile (v/v) containing 3 μM internal standards and centrifuged at 2,300 g, 4 °C for 5 min. Then phospholipids was removed by filtering the supernatant using a column (Hybrid SPE phospholipid 55261-U, Supelco, Bellefonte, PA, USA). The filtrate was desiccated and resuspended in 100 μL of 50% isopropanol in Milli-Q water (v/v) immediately before the measurement.
10
Clam Metabolite Extraction and Ultrafiltration
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Clam metabolites were extracted from 50 mg of soft tissue by addition of 1.5 mL of 50% acetonitrile solution (v/v) using a homogenizer in accordance with a previous study32 (link). The extract solution was centrifuged for 5 min at 2300 × g, 4 °C, and the supernatant was subjected to ultrafiltration.
Ultrafiltration was performed by using a centrifugal filter device (UltrafreeMC-PLHCC; Human Metabolome Technologies, Yamagata, Japan) with a regenerated cellulose membrane (molecular weight cutoff [MWCO], 5 kDa) and was carried out by using two centrifugal filter devices per sample. An aliquot of 400 μL of extract solution was subjected to ultrafiltration for 2 h at 9100 × g, 4 °C. The permeate fraction (MWCO <10 kDa) was then dried and suspended in 50 μL of Milli-Q water.
Ultrafiltration was performed by using a centrifugal filter device (UltrafreeMC-PLHCC; Human Metabolome Technologies, Yamagata, Japan) with a regenerated cellulose membrane (molecular weight cutoff [MWCO], 5 kDa) and was carried out by using two centrifugal filter devices per sample. An aliquot of 400 μL of extract solution was subjected to ultrafiltration for 2 h at 9100 × g, 4 °C. The permeate fraction (MWCO <10 kDa) was then dried and suspended in 50 μL of Milli-Q water.
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