Epididymal spermatozoa from five WT and five ChAcDel/Del mice were individually analyzed for intracellular metabolites. The spermatozoa were suspended in PBS and collected by centrifugation (500× g and 4 °C for 5 min). The cells were then treated with 800 µL of methanol and vortexed for 30 s. Next, the cell extract was treated with 550 µL of Milli-Q water containing internal standards (H3304-1002 [13 (link)], Human Metabolome Technologies, Tsuruoka, Japan) and vortexed for 30 s. The extract was obtained and centrifuged at 2300× g and 4 °C for 5 min, and then, 800 µL of the upper aqueous layer was centrifugally filtered through a Millipore 5 kDa cutoff filter at 9100× g and 4 °C for 120 min to remove proteins. The filtrate was centrifugally concentrated and resuspended in 25 µL of Milli-Q water for capillary electrophoresis time-of-flight mass spectrometry analysis. Metabolomic analysis was carried out through a facility service at Human Metabolome Technologies. For the metabolites that were able to determine the standard deviation for both groups, the Welch’s t-test was performed for statistical analysis.
H3304 1002
Manufactured by Human Metabolome Technologies
Sourced in Japan
H3304-1002 is a laboratory instrument designed for metabolomic analysis. It utilizes capillary electrophoresis coupled with mass spectrometry technology to provide comprehensive and highly sensitive detection and quantification of a wide range of metabolites in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
5 kda cutoff filter, by Merck Group (17 mentions)
Ultrafreemc plhcc, by Merck Group (11 mentions)
Milli q water, by Human Metabolome Technologies (5 mentions)
Micro smash ms 100r, by TOMY (5 mentions)
H3304 1004, by Human Metabolome Technologies (3 mentions)
Milli q water, by Merck Group (3 mentions)
Prism 6, by GraphPad (1 mentions)
Milli q, by Merck Group (1 mentions)
Ultrafree mc plhcc, by Human Metabolome Technologies (1 mentions)
Agilent 7100 ce capillary electrophoresis system, by Agilent Technologies (1 mentions)
Mass profiler professional, by Agilent Technologies (1 mentions)
Agilent 6224 tof ms, by Agilent Technologies (1 mentions)
Agilent 7100 ce system, by Agilent Technologies (1 mentions)
Insulin, by Fujifilm (1 mentions)
Human egf, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Williams e medium, by Merck Group (1 mentions)
Optima lc ms grade methanol, by Thermo Fisher Scientific (1 mentions)
Agilent g1607a ce esi ms sprayer kit, by Agilent Technologies (1 mentions)
Agilent g1603a ce ms adapter kit, by Agilent Technologies (1 mentions)
52 protocols using h3304 1002
1
Epididymal Sperm Metabolomic Analysis
2
Targeted Metabolomic Analysis of Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasma samples (500 µL) were added to 1200 µL of 1% formic acid/acetonitrile containing an internal standard solution (Solution ID: H3304-1002, Human Metabolome Technologies, Inc.) at 0°C. The solution was thoroughly mixed and centrifuged at 2300×g and 4°C for 5 minutes. The supernatant was filtered through Hybrid SPE-Phospholipid (product no. 55261-U; Sigma-Aldrich Corporation). The filtrate was desiccated and then dissolved with 80 µL of isopropanol and Milli-Q (1:1, v/v) for the LC-TOFMS analysis. The detection limit was determined at a signal-to-noise ratio>3. The candidate peaks were assigned a specific metabolite identity based on their m/z (±10 ppm) and retention time (±0.5 minutes) determined by TOFMS. The absolute concentrations were not determined, and the relative peak areas were used in the comparisons. The metabolome measurements were conducted at Human Metabolome Technologies, Inc.
3
Metabolite Quantification in Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the measurement of metabolites in the culture supernatants (CS), 20 µL of the CS and 80 µL Milli-Q (Merck KGaA, Darmstadt, Germany)−purified water containing Internal Standard Solution 1 (H3304-1002; Human Metabolome Technologies, Inc., Yamagata, Japan) were thoroughly mixed. The procedures followed those described previously.9 (link) Briefly, cationic compounds were measured in the positive mode of CE time-of-flight mass spectrometry, and anionic compounds were measured in the positive and negative modes of CE tandem MS (CE-MS/MS). Hierarchical cluster analysis (HCA) was performed by use of our proprietary software, that is, “PeakStat.” cHCECs from donors between the ages of 7 and 29 were used to obtain reproducible cultures producing high-quality cHCECs suitable for cell-injection therapy. Thus we used cHCECs from 3 donors of nearly the same age (age: 17, 14, and 18 years) to clarify the influence of the fine differences of the culture conditions. Differences between the values were statistically analyzed by use of the Student's t test or 1-way analysis of variance with Bonferroni post hoc tests (GraphPad Prism 6.0, GraphPad software). A P value <0.05 was considered statistically significant.
4
Metabolomic Analysis of Liver Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver samples were removed from the P0 pups. Frozen tissue was plunged into 50% acetonitrile solution containing internal standards (H3304-1002, Human Metabolome Technologies) at 0 °C in order to inactivate the enzymes. The tissue was then homogenized and centrifuged (2300 × g, 5 min). Subsequently, the upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter to remove proteins. The filtrate was centrifugally concentrated and resuspended in water for capillary electrophoresis coupled to mass spectrometry (CE-MS) analysis. Cationic compounds were measured in the positive mode of CE-TOFMS, and anionic compounds were measured in the positive and negative modes of CE-MS/MS as previously reported39 (link)40 (link)41 (link). Metabolome measurements and hierarchical cluster analysis were performed at a service facility of Human Metabolome Technologies, Inc.
5
Metabolite Profiling of AK4 Knockdown
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The culture medium was aspirated from a 10-cm cell culture dish and the cells were washed twice with 5 % mannitol solution (10 mL, followed by 2 mL). The cells were then treated with 800 μL methanol and left at rest for 30 s in order to inactivate enzymes. Next, the cell extract was treated with 550 μL Milli-Q water containing internal standards (H3304-1002, Human Metabolome Technologies, Inc., Tsuruoka, Japan) and left at rest for another 30 s. The extract was obtained and centrifuged at 2300 × g and 4 °C for 5 min; 800 μL of the upper aqueous layer was then centrifugally filtered through a Millipore 5-kDa cutoff filter at 9100 × g and 4 °C for 120 min to remove proteins. The filtrate was centrifugally concentrated and resuspended in 50 μL of Milli-Q water for capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) analysis. The ratios and P-values (t-test) were calculated by comparing cells transfected with control shRNA (n = 3) with those transfected with AK4 shRNA (n = 3), or cells transfected with control vector (n = 3) versus those overexpressing AK4 (n = 3). The data were normalized to the total cell numbers.
6
Quantitative Metabolomic Analysis of U2OS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U2OS cells were prepared and treated with either ox- or red-pMFc using the same procedure for monitoring the bioluminescence rhythm. As a control, 10 mM HEPES was added to the medium instead of pMFc. After 24 h incubation at 37 °C under 5% CO2, the culture medium was aspirated from a dish. Cells were washed twice with 5% mannitol and treated with 400 μL methanol. The cell extract was treated with 275 μL pure water containing internal standards (H3304-1002, Human Metabolome Technologies (HMT), Tsuruoka, Yamagata, Japan) and left to rest for another 30 s. Cell debris was removed by centrifugation at 200g at 4 °C for 5 min and 350 μL of the supernatant was filtered by centrifugation through a Millipore 5 kDa cutoff filter (Ultrafree MC-PLHCC, HMT). Filtrate was concentrated by centrifugation and re-suspended in 50 μL deionized water for metabolomic analysis at HMT.
7
Metabolome Extraction from Blood Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A blood cell suspension (200 µL) was collected by pipette and added to 1800 µL methanol containing internal standards (H3304-1002, Human Metabolome Technologies, Inc., Tsuruoka, Japan) at 0 °C to inactivate enzymes. The extract was thoroughly mixed with 2000 µL chloroform and 800 µL Milli-Q water (MilliporeSigma, Burlington, MA, USA) and centrifuged at 2300× g and 4 °C for 5 min. Next, 400 µL of the upper aqueous layer were centrifugally filtered through a Millipore 5-kDa cutoff filter to remove proteins. The filtrate was centrifugally concentrated and resuspended in 50 µL Milli-Q water for metabolome analysis.
8
Metabolite Extraction and HPLC Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
High-performance liquid chromatography (HPLC)-grade chloroform, HPLC-grade methanol (MeOH), HPLC-grade acetonitrile (ACN), HPLC-grade 2-propanol (IPA), 98% formic acid, HPLC-grade ammonium acetate, and ultrapure water were used for solvent preparation and metabolites extraction. Internal standards in extraction solvent included FFA C16:0-d3, FFA C18:0-d3, carnitine C10:0-d3, carnitine C16:0-d3, and internal standards 1 (IS1) (Human Metabolome Technologies, H3304-1002).
9
Metabolomic Analysis of CAF Secretome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze comprehensive metabolites secreted from CAF, metabolomic analyses were carried out. In brief, intracellular metabolites were extracted with methanol. Culture supernatants or cell extracts were dried in vacuum and re‐solubilized in Milli‐Q water (Merck Millipore, Billerica, MA, USA) for capillary electrophoresis‐time‐of‐flight mass spectrometry (CE‐TOFMS) analysis. Metabolite analysis using a CE‐TOFMS system25 was carried out by Human Metabolome Technologies (Tsuruoka, Japan). Concentrations of metabolites were determined from corresponding peak areas normalized to those of the internal standard (H3304‐1002; Human Metabolome Technologies) using three‐point calibration curves.
10
Metabolite Extraction and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Culture medium was aspirated from the dish, and cells were washed twice with 5% mannitol solution (10 ml and 2 ml for the first and second washes, respectively). The cells were then treated with 800 μl methanol and incubated at room temperature for 30 sec to suppress enzymatic activity. Next, 550 μl 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 sec. The cell extract was then centrifuged at 2,300 ×g for 5 min at 4°C, after which 700 μl of the supernatant was centrifugally filtered through a Millipore 5 kDa cutoff filter (UltrafreeMC-PLHCC, HMT) at 9,100 × g for 120 min at 4°C to remove macromolecules. Subsequently, the filtrate was evaporated to dryness under vacuum and reconstituted in 50 μl Milli-Q water for metabolome analysis at HMT.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!