Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis

Frontiers in Immunology

Immunology

Claudio Mauro¹  David R. Thickett¹  Aaron Scott¹  Frances S. Grudzinska¹  Alice Jasper¹  Elizabeth Sapey²  Jonathan Barlow³ 

[1] Birmingham Acute Care Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom;Birmingham Acute Care Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom;PIONEER Health Data Research- UK Hub in Acute Care, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom;Cellular Health and Metabolism Facility, School of Sport, Exercise and Rehabilitation Sciences, University of Birmingham, Birmingham, United Kingdom;

关键词: neutrophils;    extracellular flux analysis;    immunometabolism;    glycolysis;    oxidative burst;   

DOI  :  10.3389/fimmu.2023.1083072

 received in 2022-10-28, accepted in 2023-04-05,  发布年份 2023

来源: Frontiers

【 摘 要 】

Neutrophil responses are critical during inflammatory and infective events, and neutrophil dysregulation has been associated with poor patient outcomes. Immunometabolism is a rapidly growing field that has provided insights into cellular functions in health and disease. Neutrophils are highly glycolytic when activated, with inhibition of glycolysis associated with functional deficits. There is currently very limited data available assessing metabolism in neutrophils. Extracellular flux (XF) analysis assesses real time oxygen consumption and the rate of proton efflux in cells. This technology allows for the automated addition of inhibitors and stimulants to visualise the effect on metabolism. We describe optimised protocols for an XFe96 XF Analyser to (i) probe glycolysis in neutrophils under basal and stimulated conditions, (ii) probe phorbol 12-myristate 13-acetate induced oxidative burst, and (iii) highlight challenges of using XF technology to examine mitochondrial function in neutrophils. We provide an overview of how to analyze XF data and identify pitfalls of probing neutrophil metabolism with XF analysis. In summary we describe robust methods for assessing glycolysis and oxidative burst in human neutrophils and discuss the challenges around using this technique to assess mitochondrial respiration. XF technology is a powerful platform with a user-friendly interface and data analysis templates, however we suggest caution when assessing neutrophil mitochondrial respiration.

【 授权许可】

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Copyright © 2023 Grudzinska, Jasper, Sapey, Thickett, Mauro, Scott and Barlow