Metabolic profiling of LPS-activated BMDMs

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)).