Db 5ms dg

BMDMs were incubated in custom DMEM containing 10 mM U-¹³C₆ heavy labelled glucose (CLM-1396, Cambridge Isotope Laboratories) and 2 mM unlabelled glutamine and activated with 100 ng/ml LPS for 8 hours. Cells were washed three times with ice-cold saline and lysed in 80% methanol. Cell lysates were dried down using a speed-vacuum concentrator and stored at -80°C. Cellular metabolites were extracted and analysed by gas chromatography-mass spectrometry (GC-MS) using protocols described previously (52 (link), 53 (link)). Briefly, metabolite extracts were derived using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). D-myristic acid (750 ng/sample) was added as an internal standard to metabolite extracts, and metabolite abundance was expressed relative to the internal standard. GC/MS analysis was performed using an Agilent 5975C GC/MS equipped with a DB-5MS + DG (30 m × 250 µm × 0.25 µm) capillary column (Agilent J&W, Santa Clara, CA, USA). Metabolite measurements were performed at the Rosalind and Morris Goodman Cancer Research Centre Metabolomics Core Facility supported by the Canada Foundation for Innovation, The Dr. John R. and Clara M. Fraser Memorial Trust, the Terry Fox Foundation (TFF Oncometabolism Team Grand 116128) and McGill University. Mass isotopomer distribution was determined using a custom algorithm developed at McGill University (52 (link)).

Isolated CD4+ NV, EM and CM were incubated with universally heavy labelled ¹³C glucose (11.1 mM; Cambridge Isotopes) in glucose free RPMI (ThermoFisher Scientific) or ¹³C glutamine (2 mM; Cambridge Isotopes) in glutamine free (ThermoFisher Scientific). T-cells were activated with plate-bound anti-CD3 (2 μg/mL; HIT3a, BioLegend) and free anti-CD28 (20 μg/mL; CD28.2, BioLegend) for a period of either 0.5 or 4 h. Cells were then washed twice with ice-cold PBS and lysed in 80% methanol. Cell extracts were then dried down at 4 °C using a speed-vacuum concentrator.
Cellular metabolites were extracted and analysed by gas chromatography-mass spectrometry (GC-MS) using protocols described previously^{48 (link),49 (link)}. Briefly, metabolite extracts were derived using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). d-myristic acid (750 ng/sample) was added as an internal standard to metabolite extracts, and metabolite abundance was expressed relative to the internal standard. GC/MS analysis was performed using an Agilent 5975C GC/MS equipped with a DB-5MS + DG (30 m × 250 µm × 0.25 µm) capillary column (Agilent J&W, Santa Clara, CA, USA). For SITA experiments, mass isotopomer distribution was determined using a custom algorithm developed at McGill University^{48 (link)}.

The analysis was carried out at McMaster University, Ontario, Canada, on an Agilent 6890 GC equipped with a 5973 quadrupole mass spectrometer. The column used was an Agilent DB5-MS + DG, 30 m × 0.25 mm with a 0.25 μm thickness. To achieve a better separation of peaks, the initial GC temperature was set at 50 °C and held for two minutes, ramped to 200 °C at a rate of 10 °C/min and held for ten minutes, again ramped to 300 °C at a rate of 10 °C/min and held for 10 min. 1 μl of sample was introduced to the GC by splitless injection. The MS was operated in a scan mode with 12 min of sample delay, the MS quad temperature was set at 150 °C and the MS source temperature was set at 230 °C. The data acquisition was between m/z 50 and 450. Acquisition and data analysis were performed using ChemStation D.01.02 software.