Pyruvate was hyperpolarized by a method similar to that used in animal experiments. All experiments were performed on a vertical bore 7T Bruker BioSpin NMR with 10 mm broadband probe and running TopSpin3.5 software. OCI-AML3 cells (2.5 mL of a suspension in RPMI [dry powder mixed with 90% water and 10% D2O]) were added to 10-mm NMR tubes. The probe was heated to 37°C and was maintained at this temperature throughout the experiment. After auto-locking, tuning, and shimming were performed, 500 μL of the hyperpolarized compound was injected into the suspended cells using an injection port. The final concentration of hyperpolarized pyruvate in the NMR reactor was approximately 8.7 mM. Single 13C transients were taken using Waltz decoupling (zgdc pulse) every 6 seconds with a 15° flip angle. The nLac ratio was calculated as the total lactate signal divided by the sum of pyruvate and lactate signals over the first 2 minutes of the experiment. Cell viability was analyzed using trypan blue staining and counted on a BioRad TC20 automated cell counter. The percentages of viable cells were similar before and after the experiments (88–96% viable). Table 2 presents the values per million cells times 1000.
Biospin nmr
Manufactured by Bruker
The BioSpin NMR is a nuclear magnetic resonance (NMR) spectrometer designed for analytical applications in the life sciences. It provides high-resolution, multidimensional NMR spectroscopy capabilities for the analysis of biomolecules and other compounds.
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2 protocols using biospin nmr
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Hyperpolarized Pyruvate Metabolism in AML Cells
2
Natural Product Extraction and Analysis
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The culture broth was extract with double volume of EtOAc (v/v = 2:1) using Soxhlet extractor. Soxhlet extractor was then kept still to separate two layers after the mixture was shaken for 12 h at room temperature. A rotary evaporator was used to dried EtOAc fraction. The products were analyzed by HPLC-PDA using a reversed-phase column (Mightysil RP–18 GP 250–4.6 (5 μm), Kanto Chemical, Japan) at 330 nm. The binary mobile phases were include solvent A [0.05% trifluroacetic acid in HPLC-grade water] and solvent B (100% acetonitrile). The total flow rate was kept at 1 mL/min for 30 min. The percentages of solvent B used were as follows: 0–15% (0–4 min), 45% (4–10 min), 75% (10–14 min), 90% (14–20 min), 10% (20–25 min), 10% (25–30 min). The compounds were purified by preparative HPLC (Shimazu, Tokyo, Japan) with C18 column (YMC–Pack ODS-AQ (250 × 20 mm I.D., 10 μm) linked to a UV detector (330 nm). HR–QTOF ESI/MS analysis using an ACQUITY UPLC® coupled with SYNAPT G2-S (Water Corp., USA). For NMR analysis of the purified product, compounds were dried, lyophilized, and dissolved in DMSO-d6 and subjected to 700 MHz Bruker Biospin NMR for one-dimensional 1H-NMR, 13C-NMR, and two-dimensional HMBC analyses.
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