Cells were plated in fresh media and incubated for 48 h. Protein quantification was performed using proxy wells for normalization. Norvaline was used as an internal standard. Citrate and lactate standards were used to generate a standard curve for quantification. Metabolite extraction was performed using HPLC-grade ethanol (Sigma Aldrich). Samples were derivatized with methoxamine (PI45950, Thermo Fisher Scientific) and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane (Sigma Aldrich). Samples were analyzed by GC/MS using a HP-5MS Ultra Inert GC column (19091S-433UI, Agilent Technologies) installed in an Agilent 7890B gas chromatograph coupled to an Agilent 5977B mass spectrometer. Helium was used as the carrier gas. One microliter of sample was injected at 280°C. After injection, the GC oven was held at 60°C for 1 min. The oven ramped up to 320°C at 10°C/min and held for 9 min. The MS system operated under electron impact ionization mode at 70 eV, and the MS source and quadrupole were held at 230°C and 150°C, respectively. Peak abundance was determined by automated integration using MassHunter software (Agilent). Total abundance was normalized to the norvaline internal standard. Secretion rate was calculated by taking into account the specific growth rate, as determined by pre- and post-assay protein quantification.
Methoxamine
Manufactured by Thermo Fisher Scientific
Methoxamine is a laboratory reagent used in various research applications. It is a chemical compound with the formula C₁₁H₁₇NO₂. The core function of Methoxamine is to serve as a research tool for investigating biological processes and mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Hplc grade ethanol, by Merck Group (2 mentions)
Agilent 7890b gas chromatograph, by Agilent Technologies (2 mentions)
Agilent 5977b mass spectrometer, by Agilent Technologies (2 mentions)
Pi45950, by Thermo Fisher Scientific (2 mentions)
Hp 5ms ultra inert gc column, by Agilent Technologies (2 mentions)
Masshunter software, by Agilent Technologies (2 mentions)
Tri sil htp reagent, by Thermo Fisher Scientific (1 mentions)
U6 13c6 glucose, by Cambridge Isotopes (1 mentions)
3 protocols using methoxamine
1
Targeted Metabolite Profiling by GC-MS
2
Targeted Metabolite Profiling by GC-MS
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Cells were plated in fresh media and incubated for 48 h. Protein quantification was performed using proxy wells for normalization. Norvaline was used as an internal standard. Citrate and lactate standards were used to generate a standard curve for quantification. Metabolite extraction was performed using HPLC-grade ethanol (Sigma Aldrich). Samples were derivatized with methoxamine (PI45950, Thermo Fisher Scientific) and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane (Sigma Aldrich). Samples were analyzed by GC/MS using a HP-5MS Ultra Inert GC column (19091S-433UI, Agilent Technologies) installed in an Agilent 7890B gas chromatograph coupled to an Agilent 5977B mass spectrometer. Helium was used as the carrier gas. One microliter of sample was injected at 280°C. After injection, the GC oven was held at 60°C for 1 min. The oven ramped up to 320°C at 10°C/min and held for 9 min. The MS system operated under electron impact ionization mode at 70 eV, and the MS source and quadrupole were held at 230°C and 150°C, respectively. Peak abundance was determined by automated integration using MassHunter software (Agilent). Total abundance was normalized to the norvaline internal standard. Secretion rate was calculated by taking into account the specific growth rate, as determined by pre- and post-assay protein quantification.
3
Isotopic Glucose Tracing in Murine Melanoma
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B16F10 murine melanoma cells were seeded into a 60-mm dish in triplicate and incubated overnight to reach 50–60% confluence. The media was removed, and the cells were washed with PBS. Isotope-labeled glucose was dissolved to the desired concentration in pyruvate-, glucose-, and glutamine-free medium containing 10% FBS. A250 was dissolved at a 60 μg/mL concentration in complete medium containing [U6-13C6]-glucose (> 99% purity and 99% isotope enrichment for each carbon position; Cambridge Isotope Labs). The plates were incubated for 24 h in a 5% CO2 atmosphere at 37 °C. At the end of the culture period, the cells were washed with ice-cold normal saline and overlaid with 50% methanol. The cells were then detached by scraping, and the suspension was transferred to an Eppendorf tube. The samples were then snap-frozen in liquid nitrogen. After three thaw–freeze cycles, the samples were spun down at 10,000 rpm, the supernatants were transferred into new tubes, and the solvent was evaporated with a sample concentrator. The completely dried samples were derivatized with Methoxamine (Thermo) prior to silylation with Tri-Sil HTP reagent (Thermo).
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