D fructose 13c6

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.

Citric acid-d₄, L-leucine-d₁₀, D-fructose -¹³C₆, L-tryptophan-d₅, L-valine-d₃, glycerol-d₃, benzoic acid-d₅, stearic acid-d₃₅, Norvaline, HPLC grade acetone and HPLC grade methanol were all purchased from Sigma Aldrich (St. Louis, MO). L-Alanine-d₄ was purchased from Isotec (Miamisburg, OH). Optima grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ).
Two solutions were prepared to serve as internal standards using all deuterated compounds. The first solution, deuterated standard solution, contained all compounds except for stearic acid-d₃₅ at a concentration of 15 ng/μL dissolved in 50:50 MeOH:dH₂O. The second solution contained stearic acid-d₃₅ at a concentration of 60 ng/μL dissolved in acetone. A reconstitution solution was prepared as 4:1 acetonitrile:H₂O (milliQ) with Norvaline added at a concentration of 1.5 ng/μL.