CB or CB transduced BCR-ABL day 20 cells (+/- 24 hours of hypoxia) were used for NMR analyses. 20–30 million cells were quenched and the intracellular metabolites were extracted, evaporated using a SpeedVac concentrator and stored at 80°C until further analysis. For the NMR analysis dried samples were resuspended in 60 ml of 100mM sodium phosphate buffer containing 500 mM TMSP ((3-trimethylsilyl)propionic-(2,2,3,3-d4)-acid sodium salt) and 10% D2O, pH 7.0. Samples were vortexed, sonicated and centrifuged briefly, before being transferred into a 1.7mm NMR tube using an automated Gilson sample handler. One-dimensional 1D 1H-NMR spectra were acquired using a 600-MHz Bruker Avance III spectrometer (Bruker Biospin) with a TCI 1.7mm z-PFG cryogenic probe at 300 K. Each sample was automatically tuned, matched and then shimmed before acquisition of the spectrum. Spectra were processed using the MATLAB-based MetaboLab software [39 (link)]. The chemical shift was calibrated by referencing the TMSP signal to 0 ppm. Spectra were exported into Bruker format for metabolite identification and to determine concentrations using the Chenomx 7.0 software. The extracellular metabolites were measured directly in the used culture medium. All data presented here are in μMolar concentrations.
Tci 1.7mm z pfg cryogenic probe
Manufactured by Bruker
Sourced in United States
The TCI 1.7mm z-PFG cryogenic probe is a specialized laboratory equipment designed for high-resolution nuclear magnetic resonance (NMR) spectroscopy. It features a compact 1.7mm sample coil and is optimized for the detection of small sample volumes while maintaining high sensitivity. The probe incorporates a cryogenic design, allowing for efficient cooling of critical components to enhance signal-to-noise ratio and spectral quality.
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3 protocols using tci 1.7mm z pfg cryogenic probe
1
Hypoxia Impacts on BCR-ABL Metabolome
2
Fungal Metabolite Extraction for NMR Analysis
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Metabolites were extracted from 5 mg freeze-dried fungal mycelia and dried in a speed vacuum as described previously (28 (link)). Extracts were reconstituted in 50 μl of deuterated sodium phosphate buffer (100 mM, pH 7.0) containing 0.5 mM trimethylsilylpropanoic acid (TMSP), 3 mM sodium azide, and 100% D2O. Each sample was sonicated for 10 min and vortexed briefly, before a volume of 35 μl was transferred into 1.7-mm nuclear magnetic resonance (NMR) tubes.
Spectra were acquired on a Bruker 600 MHz spectrometer equipped with a TCI 1.7-mm z-PFG cryogenic probe and a Bruker SampleJet autosampler. One-dimensional (1D) 1H NMR spectra and 2D 1H-13C HSQC (heteronuclear single quantum coherence) spectra were recorded and analyzed for each sample as previously described (63 (link)).
Spectra were acquired on a Bruker 600 MHz spectrometer equipped with a TCI 1.7-mm z-PFG cryogenic probe and a Bruker SampleJet autosampler. One-dimensional (1D) 1H NMR spectra and 2D 1H-13C HSQC (heteronuclear single quantum coherence) spectra were recorded and analyzed for each sample as previously described (63 (link)).
3
Liver Metabolite Extraction and NMR Profiling
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Tissue samples from perfused livers (100 mg) were added to gentleMACs M-Tubes in cold methanol (8 μl/mg) and purified water (2 μl/mg). Tissue was homogenised (gentleMACs, Miltenyi, UK) and polar metabolites were extracted as described previously.22 (link) Samples were kept at 4°C before nuclear magnetic resonance (NMR) imaging. All spectra were acquired at 300 K on a Bruker 600 MHz spectrometer with a TCI 1.7 mm z-PFG cryogenic probe using a cooled Bruker SampleJet autosampler as previously described.22 (link) One-dimensional 1H-NMR spectra were processed using the NMRlab and Metabolab programmes within Matlab, version R2016b (MathWorks, MA, USA). Two-dimensional heteronuclear single quantum coherence (HSQC) spectra processing was initially performed using NMRPipe23 (link) with the Hyberts extension for processing non-uniformity sampling spectra24 (link) and subsequent analysis was performed using NMRLab in MATLAB_R2016b (The Mathworks). Cosine-squared window functions were applied to both dimensions and spectra were phased manually. Calibration was carried out manually using L-lactic acid as a reference peak (δ 1.31/22.9 ppm) and scaling was performed using TSA-scaling factors from 1D NOESY (Nuclear Overhauser Effect Spectroscopy) associated spectra. Peak identification used Metabolab25 with reference to HMDB (Human Metabolome Database : https://hmdb.ca).
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