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Protocol for stable isotope tracing of primary human peripheral blood mononuclear cells
Samantha N Hart1, Holden Williams2, William B Inabnet3
1Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, 741 S Limestone, Lexington, KY 40536, USA; Barnstable Brown Diabetes and Obesity Research Center, University of Kentucky, 1000 S Limestone, Lexington, KY 40536, USA.
Abstract:
Stable isotope tracing gives direct insight into the rate of metabolic reactions occurring in cells by analyzing the incorporation of labeled carbons into metabolic pathways. Here, we present a protocol for stable isotope tracing of primary human peripheral blood mononuclear cells (PBMCs). We describe the steps for culturing and exposing PBMCs to stable isotopes U13C glucose and U13C glutamine. We then detail procedures for derivatization, metabolite purification, and tracing analysis via single-quadrupole gas chromatography-mass spectrometry (GC-MS).
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