ORIGINAL RESEARCH article
Front. Immunol.
Sec. Systems Immunology
Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1676550
Comprehensive Real-Time Metabolic Profiling of Peripheral Blood Mononuclear Cells Reveals Important Methodological Considerations for Immunometabolism Research
Provisionally accepted
- 1University of Birmingham, Birmingham, United Kingdom
- 2Universitas Tadulako, Palu, Indonesia
Select one of your emails
You have multiple emails registered with Frontiers:
Notify me on publication
Please enter your email address:
If you already have an account, please login
You don't have a Frontiers account ? You can register here
The utility of measuring real-time cellular bioenergetics of peripheral blood mononuclear cells (PBMCs) as biomarkers in disease monitoring, such as the bioenergetic health index, are of emerging interest. However, various experimental factors can impact the accuracy and reproducibility of these measurements. This study aimed to explore key experimental variables including injection strategy, blood processing time, and isolation method to improve the reliability and biological relevance of metabolic assessments using PBMCs. PBMC bioenergetics were probed in real-time using extracellular flux analysis to identify optimal seeding density and injection protocol. Using a modified protocol, we assessed to what extent blood processing time and/or isolation method (SepMate™ vs. EasySep™ Direct) influences PBMC bioenergetics under basal and stimulated conditions. Advanced metabolic control analysis including mitochondrial and glycolytic ATP supply flux, respiratory control ratio, bioenergetic health index, and mitochondrial toxicity index were used to identify and quantify PBMC bioenergetics. We show that measures of metabolic profiling such as mitochondrial respiration, glycolytic activity, ATP supply flux, and respiratory control ratio are significantly diminished in PBMCs due to blood processing delay (48-72 hours) and are influenced by isolation method. We also demonstrate that extended blood processing time significantly lowers T cell activation capacity in PBMCs, evidenced by decreased responses of mitochondrial and glycolytic ATP supply to CD3/CD28 activation. This study underscores the importance of optimising experimental protocols for PBMC metabolic profiling and indicates that bioenergetic metrics, such as the bioenergetic health index, are sensitive to blood processing delays and/or isolation method.
Keywords: PBMCs, Bioenergetics, Extracellular flux analysis, Mitochondria, Glycolysis, metabolic profiling, blood processing time
Received: 30 Jul 2025; Accepted: 06 Oct 2025.
Copyright: © 2025 Barlow, MacLeod, Pradana and Wadley. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Jonathan Barlow, j.p.barlow@bham.ac.uk
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.