Intracellular Metabolite Profiling of Cancer Cells

After removing the media, cells were washed with 0.9% NaCI and quenched with 100% cold methanol, followed by the addition of isovolumetric water. We scraped the cells thoroughly and transferred into an Eppendorf tube containing 400 μL chloroform. Next, cells were vibrated at 1400 rpm for 20 min, followed by centrifugation at 16,100 × g for 5 min. Samples were then dried at a low temperature under vacuum to avoid metabolite degradation. The extracted intracellular metabolites were analyzed by gas chromatography and time-of-flight mass spectrometry, provided by BioMaker (China) as a service. We determined glycolytic capacity of cancer cells by using the Glucose Uptake Colorimetric Assay Kit (Biovision), Lactate Colorimetric Assay Kit (Biovision), and ATP Assay Kit (Promega), according to the manufacturers’ instructions. Glucose-induced ECAR was monitored by the Seahorse XF24 Flux Analyzer as previously described [34 (link)]. Cells were first seeded in a XF24 well plate and cultured overnight. Then the cells were washed with Seahorse buffer, followed by sequentially injecting 10 mM glucose, 1 µM oligomycin, and 80 mM 2-deoxyglucose. ECAR measurements were normalized to the number of cells per well.