Before extraction, samples were moved from −80 °C storage to wet ice and thawed. Extraction buffer consisting of 1% formic acid (Millipore Sigma) and 4.5 µM metabolomics amino acid mix standard (Cambridge Isotope Laboratories, Inc.), was prepared and chilled on dry ice. A cocktail of polyamine standards containing 10 µM Spermine, 10 µM SPD, 10 µM N-acetylspermine and 10 µM Putrescine was prepared in advance of the extraction (Sigma Aldrich). The samples and cocktail were extracted by mixing 10 µl of sample with 10 µl of extraction buffer in 2 ml liquid chromatography (LC) vials containing a 250 µl glass insert. All vials were then incubated at 60 °C in a heat-block for 30 min, followed by addition of 80 µl of warm (60 °C) 100% methanol to the individual samples. The glass inserts were then transferred to microfuge carrier tubes and centrifuged at 3,000g for 20 min at 4 °C. Supernatant (20 µl) was then transferred to a new LC vial containing a new glass insert for LC–MS analysis and the remaining sample was placed in −80 °C for long-term storage.
Metabolomics amino acid mix standard
Manufactured by Cambridge Isotopes
The Metabolomics amino acid mix standard is a laboratory product designed for use in metabolomics research. It contains a mixture of amino acids that can be used as a reference standard for the identification and quantification of these compounds in biological samples. The standard is formulated to provide a comprehensive set of amino acids commonly found in metabolomic studies.
Automatically generated - may contain errors
Lab products found in correlation
Speedvac, by Thermo Fisher Scientific (3 mentions)
Optima lc ms grade water, by Thermo Fisher Scientific (3 mentions)
Methanol, by Thermo Fisher Scientific (3 mentions)
Formic acid, by Merck Group (2 mentions)
Disruption beads, by Research Products International (2 mentions)
Beadblaster homogenizer, by Benchmark Scientific (2 mentions)
Tracefinder 4, by Thermo Fisher Scientific (2 mentions)
Xcalibur quanbrowser 2, by Thermo Fisher Scientific (2 mentions)
Hplc grade chloroform, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
D7 sphinganine, by Avanti Polar Lipids (1 mentions)
8 protocols using metabolomics amino acid mix standard
1
Metabolomics Extraction Protocol for Polyamines
2
Metabolite Extraction and Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to extraction, samples were moved from −80°C storage to wet ice and thawed. Extraction buffer, consisting of 80% methanol (Cat# 615130025, Thermo Fisher) and 500 nM metabolomics amino acid mix standard (Cat# MSK-A2–1.2, Cambridge Isotope Laboratories), was prepared and placed on dry ice. Samples were extracted by mixing 50 μL of sample with 950 μL of extraction buffer in 2.0 mL screw cap vials containing ~100 μL of disruption beads (Cat# 9835, Research Products International). Each sample was homogenized for 10 cycles on a BeadBlaster homogenizer (Benchmark Scientific). Cycling consisted of a 30 s homogenization time at 6 m/s followed by a 30 s pause. Samples were subsequently spun at 21,000 rcf for 3 min at 4°C. A set volume of each (450 μL) was transferred to a 1.5 mL tube and dried down by SpeedVac (Thermo Fisher). Samples were reconstituted in 50 μL of Optima LC/MS grade water (Cat# W6500, Fisher Scientific). Samples were sonicated for 2 min, then spun at 21,000 rcf for 3 min at 4°C. Twenty μL were transferred to LC vials containing glass inserts for analysis. The remaining sample was placed in −80°C for long term storage.
3
Targeted Lipidomics and Metabolomics Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HPLC grade chloroform (Sigma-Aldrich, 650498-1 L) and methanol (Sigma-Aldrich, 34860-1 L-R) were obtained from Sigma-Aldrich. HPLC grade water (W5-1) was from Fisher Scientific. The following internal standards were used in targeted lipidomics analyses: Cer/Sph mixture II (contains Cer(d18:1/12:0), cat #LM6005, Avanti Polar Lipids), d 7-sphinganine (860658, Avanti Polar Lipids), d 3-deoxysphinganine (860474, Avanti Polar Lipids), C12-doxCer [Cer(m18:1/12:0)] (860455P-1 mg, Avanti Polar Lipids), and C12-dihydro-doxCer [Cer(m18:0/12:0)] (860481P-1 mg, Avanti Polar Lipids). The following internal standards were used in metabolomics analyses: d 3-serine (DLM-582-0.1, Cambridge Isotope Laboratories) and the Metabolomics Amino Acid Mix Standard (contains 13C3, 15n -serine, cat #MSK-A2-1.2, Cambridge Isotope Laboratories).
4
Quantification of Polar Metabolites in A549 Cells
5
Metabolomics Extraction and Sample Prep
6
Targeted Metabolomics of Macrophages Treated with LDL
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Targeted metabolomics was performed on BMDMs treated with agLDL (50 µg/mL), oxLDL (50 µg/mL), or vehicle for 24 h by the NYU Metabolomics Core Resource Laboratory, as described (51 (link)). Briefly, metabolites were extracted using 80% methanol and a metabolomics amino acid mix standard (Cambridge Isotope Laboratories Inc.), homogenized, centrifuged, dried by speedvac (Thermo Fisher Scientific), and reconstituted in Optima LC/MS grade water (Thermo Fisher Scientific). Samples were sonicated and subjected to LC/MS analysis.
7
Polar Metabolite Profiling of A549 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 cells were infected with lentiviruses containing either CTRL or UGDH-g1 or UXS1 g1in biological triplicates. Cells were selected with Puromycin for five days. Cells at indicated time-points were washed thrice with ice-cold PBS and extracted on dry ice on 1ml 80% methanol containing 500nM internal standards (Metabolomics Amino Acid Mix Standard; Cambridge Isotope Laboratories). Cell extracts were collected using a cell scraper and transferred to a microcentrifuge tube. Samples were vortexed for 15 minutes at 4°C and centrifuged at 18000 x g for 10 minutes at 4°C. Supernatants were transferred to a new microcentrifuge tube and stored at -80°C until analysis. These samples were then dry evaporated using vacuum centrifugation. Polar metabolite profiling was performed on dried polar extracts at the Whitehead metabolite profiling core facility. It was performed on a QExactive orbitrap mass spectrometer equipped with an ion Max source and a HESI II probe coupled with a Dionex Ultimate 3000 HPLC system containing SeQuant ® ZIC ® -pHILIC analytical column. Relative quantitation of polar metabolites, including UDPGA, was performed with XCalibur QuanBrowser 2.2 and TraceFinder 4.1 (both Thermo Fischer Scientific) using a 5ppm mass tolerance and referencing an in-house library of chemical standards.
8
Metabolite Extraction from Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed twice with ice cold PBS and extracted on dry-ice in 0.8 mL 80% methanol containing 500 nM internal standards (Metabolomics Amino Acid Mix Standard: Cambridge Isotope Laboratories, Inc.). Cell extracts were collected using a cell scraper and transferred to a microcentrifuge tube. Samples were vortexed for 10 minutes at 4°C and centrifuged at 17,000g for 10 minutes at 4°C. Supernatants were transferred to new microcentrifuge tubes and evaporated to dryness by vacuum centrifugation. Dried polar extracts were stored at -80°C until analysis.
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!