Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis
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 glycol...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1083072/full |
| _version_ | 1797840007074938880 |
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| author | Frances S. Grudzinska Alice Jasper Elizabeth Sapey Elizabeth Sapey David R. Thickett Claudio Mauro Aaron Scott Jonathan Barlow |
| author_facet | Frances S. Grudzinska Alice Jasper Elizabeth Sapey Elizabeth Sapey David R. Thickett Claudio Mauro Aaron Scott Jonathan Barlow |
| author_sort | Frances S. Grudzinska |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-09T16:07:24Z |
| format | Article |
| id | doaj.art-59086772f925458b814c28481acc056c |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-04-09T16:07:24Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-59086772f925458b814c28481acc056c2023-04-25T04:45:49ZengFrontiers Media S.A.Frontiers in Immunology1664-32242023-04-011410.3389/fimmu.2023.10830721083072Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysisFrances S. Grudzinska0Alice Jasper1Elizabeth Sapey2Elizabeth Sapey3David R. Thickett4Claudio Mauro5Aaron Scott6Jonathan Barlow7Birmingham Acute Care Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United KingdomBirmingham Acute Care Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United KingdomBirmingham Acute Care Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United KingdomPIONEER Health Data Research- UK Hub in Acute Care, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United KingdomBirmingham Acute Care Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United KingdomBirmingham Acute Care Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United KingdomBirmingham Acute Care Research Group, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, United KingdomCellular Health and Metabolism Facility, School of Sport, Exercise and Rehabilitation Sciences, University of Birmingham, Birmingham, United KingdomNeutrophil 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.https://www.frontiersin.org/articles/10.3389/fimmu.2023.1083072/fullneutrophilsextracellular flux analysisimmunometabolismglycolysisoxidative burst |
| spellingShingle | Frances S. Grudzinska Alice Jasper Elizabeth Sapey Elizabeth Sapey David R. Thickett Claudio Mauro Aaron Scott Jonathan Barlow Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis Frontiers in Immunology neutrophils extracellular flux analysis immunometabolism glycolysis oxidative burst |
| title | Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis |
| title_full | Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis |
| title_fullStr | Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis |
| title_full_unstemmed | Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis |
| title_short | Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis |
| title_sort | real time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis |
| topic | neutrophils extracellular flux analysis immunometabolism glycolysis oxidative burst |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1083072/full |
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