Purine metabolism assay was performed as described previously [10 (link)]. Briefly, cells were cultured in RPMI 1640 at a density of 1 × 106 cells per ml, after addition [13C5,15N4]-hypoxanthine (Cambridge Isotope Laboratories, cat. No. CNLM-7894-PK), cells were cultured for an additional 4 h, then harvested and pelleted. The reaction was quenched in cold 80% methanol, cells were centrifuged at 15,000 r.p.m. for 15 min and metabolites (IMP, AICAR, Inosine and HX) in the supernatant were analyzed by LC-MS [17 (link)]. IMP synthesis through the purine salvage pathway was measured by 13C5,15N4 incorporation (molecular weight peak IMP-labeled); The relative concentrations were defined according to the standard curve of compounds dissolved in 80% methanol without correcting for cell matrix effect.
13c5 15n4 hypoxanthine
Manufactured by Cambridge Isotopes
[13C5,15N4]-hypoxanthine is a stable isotope-labeled compound. It contains five carbon atoms and four nitrogen atoms that are isotopically labeled. This compound can be used as a research tool in various scientific applications.
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2 protocols using 13c5 15n4 hypoxanthine
1
Purine Metabolism Assay Protocol
2
Tracing Metabolite Flux in Purine Synthesis
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Metabolite flux assays were performed as described previously.12 (link) Cells were cultured in RPMI 1640 media at a density of 5 × 105/mL. Isotope-labeled [13C2, 15N] Glycine (Sigma, Cat#489522) or [13C5, 15N4] Hypoxanthine (Cambridge Isotope Laboratories. Tewksbury, MA, US, Cat#CNLM-7894-PK) was added to cells then cultured for 2 hours. Cells were then harvested, pelleted and quenched in cold 80% methanol, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was applied for metabolite analysis by AB SciexQtrap 5500 coupled with Waters Acquity UPLC. IMP synthesis (flux) through de novo purine synthesis pathway was measured by [13C2, 15N] incorporation into cells (molecular weight peak IMP+3); IMP synthesis (flux) through purine salvage pathway measured by [13C5, 15N4] incorporation into cells (molecular weight peak IMP+9).
For PRPP measurement, cells were cultured in RPMI 1640 media and then labeled with [U-13C6] D-glucose (Cambridge isotope laboratories) for 5 minutes. Cells were then harvested, pelleted and quenched in cold 80% methanol, the newly synthesized PRPP in cells were measured by [13C5] incorporation into cells (molecular weight peak PRPP+5). For ADP and GDP feedback inhibition test, we first treated cell by 2 mM ADP or 1.5 mM GDP for 24 hours, and then harvested the cells and measured the newly synthesized.
For PRPP measurement, cells were cultured in RPMI 1640 media and then labeled with [U-13C6] D-glucose (Cambridge isotope laboratories) for 5 minutes. Cells were then harvested, pelleted and quenched in cold 80% methanol, the newly synthesized PRPP in cells were measured by [13C5] incorporation into cells (molecular weight peak PRPP+5). For ADP and GDP feedback inhibition test, we first treated cell by 2 mM ADP or 1.5 mM GDP for 24 hours, and then harvested the cells and measured the newly synthesized.
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