Isotopic Tracer Analysis of Macrophage Metabolism

¹³C₆-glucose was purchased from Cambridge Isotope Laboratories and d-fructose-¹³C₆ (587621) was purchased from Sigma-Aldrich. SITA with ¹³C-glucose and ¹³C-fructose allowed for the identification of isotopomer distribution of metabolites. Both ¹³C fructose and glucose were added in glucose-free Dulbecco’s modified Eagle’s medium supplemented with 10% dialysed FBS and 50 µM β-mercaptoethanol in the presence of LPS (10 ng/mL). Metabolites were extracted after 24 h of LPS activation and analysed by liquid chromatography-mass spectrometry (LC-MS) using methods previously described^{48 (link)}. In brief, 1 × 10⁶ BMDMs were washed with cold PBS and metabolites were extracted with 200 µL of ice-cold extraction buffer (methanol, acetonitrile and water (50:30:20)). Samples were centrifuged at 21.1 × g for 10 min at 4 °C and the supernatant was collected for LC-MS analysis. Extracellular metabolites were extracted using 10 µL of culture media added to 490 μL of ice-cold extraction buffer and centrifuged at the aforementioned speed. Supernatants were collected and run through LC-MS analysis. LC-MS machine information and operation is further described in Labuschagne et al.^{49 (link)}. Spectra analysis was performed using the Thermo TraceFinder software.