For gas chromatography-tandem mass spectrometry (GC-MS), subcutaneous tumor fragments weighing 5–15 mg were homogenized using an electronic tissue disruptor (Qiagen) in ice-cold 80:20 methanol:water (vol:vol) followed by with three freeze-thaw cycles in liquid nitrogen. The supernatant was collected after a 10 min centrifugation at 13,000xg at 4°C then lyophilized. To analyze isotope enrichment in the plasma, whole blood was chilled on ice then centrifuged for 1 minute at 13,000xg at 4°C to separate the plasma. Aliquots of 20–40 μl of plasma were added to 80:20 methanol:water to extract the metabolites, then lyophilized using a SpeedVac (Thermo), and re-suspended in 40 μl anhydrous pyridine. This solution was added to pre-prepared GC-MS autoinjector vials containing 80 μl N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) to derivatize polar metabolites. The samples were incubated at 70°C for 1 hour, then aliquots of 1 μl were injected for analysis. Samples were analyzed using either an Agilent 6890 or an Agilent 7890 gas chromatograph coupled to an Agilent 5973N or 5975C Mass Selective Detector, respectively. The observed distributions of mass isotopologues were corrected for natural abundance39 (link).
Electronic tissue disruptor
Manufactured by Qiagen
The electronic tissue disruptor is a lab equipment product designed to efficiently homogenize and disrupt various types of tissue samples. Its core function is to provide a controlled and consistent disruption of tissue samples for further processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Speedvac, by Thermo Fisher Scientific (3 mentions)
Lc ms grade, by Thermo Fisher Scientific (3 mentions)
5975c mass selective detector, by Agilent Technologies (2 mentions)
Agilent 6890, by Agilent Technologies (2 mentions)
Agilent 5973n, by Agilent Technologies (2 mentions)
Agilent 7890, by Agilent Technologies (2 mentions)
Ivis spectrum, by PerkinElmer (1 mentions)
5 protocols using electronic tissue disruptor
1
GC-MS Analysis of Tumor Metabolites
2
GC-MS Analysis of Tumor Metabolites
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For gas chromatography-tandem mass spectrometry (GC-MS), subcutaneous tumor fragments weighing 5–15 mg were homogenized using an electronic tissue disruptor (Qiagen) in ice-cold 80:20 methanol:water (vol:vol) followed by with three freeze-thaw cycles in liquid nitrogen. The supernatant was collected after a 10 min centrifugation at 13,000xg at 4°C then lyophilized. To analyze isotope enrichment in the plasma, whole blood was chilled on ice then centrifuged for 1 minute at 13,000xg at 4°C to separate the plasma. Aliquots of 20–40 μl of plasma were added to 80:20 methanol:water to extract the metabolites, then lyophilized using a SpeedVac (Thermo), and re-suspended in 40 μl anhydrous pyridine. This solution was added to pre-prepared GC-MS autoinjector vials containing 80 μl N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) to derivatize polar metabolites. The samples were incubated at 70°C for 1 hour, then aliquots of 1 μl were injected for analysis. Samples were analyzed using either an Agilent 6890 or an Agilent 7890 gas chromatograph coupled to an Agilent 5973N or 5975C Mass Selective Detector, respectively. The observed distributions of mass isotopologues were corrected for natural abundance39 (link).
3
Bioluminescence Imaging of Brain Metastases
4
Extracting Metastatic Brain Tumor Metabolites
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Based on the observed signal approximate the region of metastatic lesion in the brain. Resect BLI+ brain metastatic lesion using surgical blades and verify BLI signal in the resected metastatic lesion using IVIS imager. See
Flash freeze collected tissues in liquid nitrogen and store at −80°C.
Homogenize 30–50 mg of metastatic lesion using an electronic tissue disruptor (Qiagen) in 1 mL chilled 80% methanol (LC-MS grade, Fisher Scientific). See
Subject the homogenized solution three freeze-thaw cycles using liquid nitrogen and 37°C water bath.
Centrifuge samples at 13,000 g for 10 min to precipitate macromolecules.
Collect supernatant carefully and lyophilize using Speed-Vac ( Schematic illustration showing metabolite extraction and analysis procedures
5
Metabolite Analysis of Brain Metastasis
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For in vivo steady state metabolite analysis, once the average brain only or whole-body photon flux reached 106–107 p/sec/cm2/sr, mice were subjected to cardiac perfusion post D-luciferin injection and brain was isolated as described earlier.
Use ex vivo brain BLI signal to mark and surgically remove metastatic lesions.
Flash freeze tumors in liquid nitrogen and store at −80°C until further processing.
Sample preparation for Mass spectroscopy. Homogenize metastatic lesions (30–50 mg) using an electronic tissue disruptor (Qiagen) in 1 mL chilled 80% methanol (LC-MS grade, Fisher Scientific). Next, follow three freeze-thaw cycles by using liquid nitrogen and a 37°C water bath. Centrifuge samples at 13,000 g for 10 min at 4°C. Collect supernatant and lyophilize using a Speed-Vac.
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