Protocol for stable isotope tracing of primary human peripheral blood mononuclear cells

Authors

• Samantha N. Hart, University of KentuckyFollow
• Holden C. Williams, University of KentuckyFollow
• William B. Inabnet III, University of Kentucky
• Joshua P. Steiner, University of Kentucky
• Varun Jain, University of Kentucky
• Philip A. Kern, University of KentuckyFollow
• Cecily R. Wood, University of KentuckyFollow
• Barbara S. Nikolajczyk, University of KentuckyFollow
• Lance A. Johnson, University of KentuckyFollow

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).

Document Type

Article

Publication Date

2026

Notes/Citation Information

© 2026 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 

Digital Object Identifier (DOI)

https://doi.org/10.1016/j.xpro.2026.104450

Funding Information

This publication was made possible by the University of Kentucky CNS Metabolism (CNS-Met) COBRE, supported by a grant from the National Institute of General Medical Sciences, NIGMS (P20 GM148326), National Institutes of Health. Funding for personnel was also provided by training grant UL1TR001997. Funding for this work was also provided by the Diabetes Research Center at Washington University, NIH P30 DK020579. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institutes of Health. Graphical abstract was created with BioRender.com.

Repository Citation

Hart, Samantha N.; Williams, Holden C.; Inabnet, William B. III; Steiner, Joshua P.; Jain, Varun; Kern, Philip A.; Wood, Cecily R.; Nikolajczyk, Barbara S.; and Johnson, Lance A., "Protocol for stable isotope tracing of primary human peripheral blood mononuclear cells" (2026). Molecular and Cellular Biochemistry Faculty Publications. 210.
https://uknowledge.uky.edu/biochem_facpub/210