Metabolic Analysis of Activated T-Cells

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)}.