Name: Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils (Video) | JoVE Journal
Uploaded: 2023-06-02T13:00:00.000+00:00
Duration: 9 min 39 s
Description: Learn to quantify mitochondrial respiration in mouse and human neutrophils and HL60 cells using a metabolic extracellular flux analyzer. This method enables real-time bioenergetic profiling under normal and disease conditions.

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We acknowledge Dr. Anthony T. Vella and Dr. Federica Aglianoin from the Department of Immunology at UConn Health for their training in using the metabolic extracellular flux analyzer, and Dr. Lynn Puddington in the Department of Immunology at UConn Health for her support of the instruments. We acknowledge Dr. Geneva Hargis from UConn School of Medicine for her help with scientific writing and editing of this manuscript. This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (R01HL145454), National Institute of General Medical Sciences (R35GM147713 and P20GM139763), a startup fund from UConn Health, and a Career re-entry fellowship from the American Association of Immunologists.

The authors declare no competing financial interest.

The standard procedure that measures the mitochondrial respiration of neutrophils using the metabolic extracellular flux analyzer is limited by many factors, including cell number, cell growth, and viability. Each compound concentration varies among the type and source of cells in this assay.&#160;Oligomycin and rotenone/antimycin A are mostly used in a similar concentration among most cell types. However, as the FCCP-induced maximum respiratory rate varies among different cells, careful titration of FCCP is required to ...

Mitochondria play a major role in cell bioenergetics, which generates adenosine triphosphate (ATP) by oxidative phosphorylation (OXPHOS). In addition to this, the role of mitochondria extends into the generation and detoxification of reactive oxygen species, cytoplasmic and mitochondrial matrix calcium regulation, cellular synthesis, catabolism, and the transport of metabolites within the cell1. Mitochondrial respiration is essential in all cells, as their dysfunction can result in metabolic problems2, including cardiovascular diseases3 and a wide variety of neurodegenerative diseases, such as age-related macular degeneration4, Parkinson&#39;s and Alzheimer&#39;s diseases5, and Charcot-Marie-Tooth disease 2 A (CMT2A)6.
Electron microscopic studies on neutrophils revealed there are relatively few mitochondria7, and they rely heavily on glycolysis for their energy production as mitochondrial respiration rates are very low8. However, mitochondria are crucial for neutrophil functions, such as chemotaxis9 and apoptosis10,11,12. A previous study revealed a complex mitochondrial network in human neutrophils with high membrane potential. The mitochondrial membrane potential loss is an early indicator of neutrophil apoptosis10. Treatment with mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showed significant inhibition in chemotaxis, along with a change in mitochondrial morphology9,10.
Although the primary energy source for neutrophils is glycolysis, mitochondria provide the ATP that initiates neutrophil activation by fueling the first phase of purinergic signaling, which boosts Ca2+ signaling, amplifies mitochondrial ATP production, and initiates neutrophil functional responses13. Dysfunction of the mitochondrial respiratory chain results in excessive production of toxic reactive oxygen species (ROS) and leads to pathogenic damages14,15,16. NETosis, which is the process of forming neutrophil extracellular traps (NETs), is a critical property of neutrophils that helps them fight against pathogens17 and contributes to many pathological conditions, including cancer, thrombosis, and autoimmune disorders18. Mitochondrial-derived ROS contribute to NETosis19, mitochondrial DNA can be a component of NETs18, and altered mitochondrial homeostasis impairs NETosis20,21,22,23,24. Furthermore, during normal differentiation or maturation, neutrophil metabolic reprogramming gets reversed by limiting glycolytic activity, and they engage in mitochondrial respiration and mobilize intracellular lipids25,26.
The metabolic extracellular flux analyzer can continuously monitor and quantify live cell mitochondrial respiration and glycolysis. The analyzer utilizes a 96-well plate format sensor cartridge and two fluorophores to quantify oxygen (O2) concentration and pH changes. The sensor cartridge is above the cell monolayer during the assay and forms a ~200 nm high microchamber. The optical fiber bundles in the analyzer are used to excite the fluorophores and detect the fluorescent intensity changes. Real-time changes in O2 concentration and pH are automatically calculated and shown as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). There are four ports on the sensor cartridge that allow loading up to four compounds into each well during the assay measurements. This protocol focuses on quantifying the mitochondrial respiration of mouse and human neutrophils, as well as the neutrophil-like HL60 cells, using the metabolic extracellular flux analyzer.

<table><tbody><tr><td>37 &amp;deg;C non-CO&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;incubator</td><td>Precision</td><td>Economy Model 2EG</td><td>Instrument</td></tr><tr><td>Biorender</td><td></td><td></td><td>Software Application</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Model 5810R</td><td>Instrument</td></tr><tr><td>Corning&amp;nbsp;Cell-Tak Cell and Tissue Adhesive</td><td>Corning</td><td>102416-100</td><td>Reagent</td></tr><tr><td>EasySep Magnet</td><td>STEMCELL</td><td>18000</td><td>Magnet</td></tr><tr><td>EasySepMouse Neutrophil Enrichment kit</td><td>STEMCELL</td><td>19762A</td><td>Reagents</td></tr><tr><td>Graphpad Prism 9</td><td></td><td></td><td>Software Application</td></tr><tr><td>Human Serum Albumin Solution (25%)</td><td>GeminiBio</td><td>800-120</td><td>Reagents</td></tr><tr><td>Ketamine (VetaKet)</td><td>DAILYMED</td><td>NDC 59399-114-10</td><td>Anesthetic</td></tr><tr><td>PBS</td><td>Cytiva</td><td>SH30256.01</td><td>Reagents</td></tr><tr><td>Plate buckets</td><td>Eppendorf</td><td>UL155</td><td>Accessory</td></tr><tr><td>PolymorphPrep</td><td>PROGEN</td><td>1895 (previous 1114683)</td><td>polysaccharide solution</td></tr><tr><td>Purified mouse anti-human CD18 antibody</td><td>Biolegend</td><td>302102</td><td>Clone TS1/18</td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>11-875-093</td><td>Reagents</td></tr><tr><td>Seahorse metabolic extracellular flux analyzer</td><td>Agilent</td><td>XFe96</td><td>Instrument</td></tr><tr><td>Seahorse XF Cell Mito Stress Test Kit</td><td>Agilent</td><td>103015-100</td><td>mitochondrial stress test Kit</td></tr><tr><td>Swing-bucket rotor</td><td>Eppendorf</td><td>A-4-62</td><td>Rotor</td></tr><tr><td>Vactrap 2 Vacum Trap</td><td>Fox Lifesciences</td><td>3052101-FLS</td><td>Instrument</td></tr><tr><td>Wave</td><td></td><td></td><td>Software Application</td></tr><tr><td>XF 1.0 M Glucose Solution</td><td>Agilent</td><td>103577-100</td><td>Reagent</td></tr><tr><td>XF 100 mM Pyruvate Solution</td><td>Agilent</td><td>103578-100</td><td>Reagent</td></tr><tr><td>XF 200 mM Glutamine Solution</td><td>Agilent</td><td>103579-100</td><td>Reagent</td></tr><tr><td>XF DMEM medium</td><td>Agilent</td><td>103575-100</td><td>Reagent</td></tr><tr><td>XFe96 FluxPak</td><td>Agilent</td><td>102601-100</td><td>Material</td></tr><tr><td>Xylazine (AnaSed Injection)</td><td>DAILYMED</td><td>NDC 59399-110-20</td><td>Anesthetic</td></tr></tbody></table>

real-time measurement of the mitochondrial bioenergetic profile of neutrophils

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

Representative OCR dynamics are shown indicating the mitochondrial respiration changes in response to oligomycin, FCCP, and rotenone/antimycin A mixture of mouse neutrophils (Figure 3A), human neutrophils (Figure 3B), and undifferentiated and differentiated HL60 cells (Figure 3C). In all cells, oligomycin treatment decreases the OCR value by inhibiting the proton channel of ATP synthase; FCCP treatment restores the OCR value by incr...

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

real-time-measurement-mitochondrial-bioenergetic-profile

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Learn to quantify mitochondrial respiration in mouse and human neutrophils and HL60 cells using a metabolic extracellular flux analyzer. This method enables real-time bioenergetic profiling under normal and disease conditions.

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

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