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.

Heparinized whole-blood samples were obtained from healthy human donors after obtaining informed consent, as approved by the Institutional Review Board of UConn Health in accordance with the Declaration of Helsinki. All animal experiments followed the UConn Health Institutional Animal Care and Use Committee (IACUC) guidelines, and approval for the use of rodents was obtained from the UConn Health IACUC according to criteria outlined in the Guide for the Care and Use of Laboratory Animals from the National Institutes of H...

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.

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			<td>37 &#176;C non-CO2&#160;incubator</td>
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			<td>Economy Model 2EG</td>
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		<tr>
			<td>Biorender</td>
			<td></td>
			<td></td>
			<td>Software Application</td>
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		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Model 5810R</td>
			<td>Instrument</td>
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		<tr>
			<td>Corning&#160;Cell-Tak Cell and Tissue Adhesive</td>
			<td>Corning</td>
			<td>102416-100</td>
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		<tr>
			<td>EasySep Magnet</td>
			<td>STEMCELL</td>
			<td>18000</td>
			<td>Magnet</td>
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		<tr>
			<td>EasySepMouse Neutrophil Enrichment kit</td>
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			<td>19762A</td>
			<td>Reagents</td>
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		<tr>
			<td>Graphpad Prism 9</td>
			<td></td>
			<td></td>
			<td>Software Application</td>
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		<tr>
			<td>Human Serum Albumin Solution (25%)</td>
			<td>GeminiBio</td>
			<td>800-120</td>
			<td>Reagents</td>
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		<tr>
			<td>Ketamine (VetaKet)</td>
			<td>DAILYMED</td>
			<td>NDC 59399-114-10</td>
			<td>Anesthetic</td>
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		<tr>
			<td>PBS</td>
			<td>Cytiva</td>
			<td>SH30256.01</td>
			<td>Reagents</td>
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		<tr>
			<td>Plate buckets</td>
			<td>Eppendorf</td>
			<td>UL155</td>
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			<td>PolymorphPrep</td>
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			<td>1895 (previous 1114683)</td>
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			<td>Purified mouse anti-human CD18 antibody</td>
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			<td>Gibco</td>
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		<tr>
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			<td>Agilent</td>
			<td>XFe96</td>
			<td>Instrument</td>
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		<tr>
			<td>Seahorse XF Cell Mito Stress Test Kit</td>
			<td>Agilent</td>
			<td>103015-100</td>
			<td>mitochondrial stress test Kit</td>
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		<tr>
			<td>Swing-bucket rotor</td>
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			<td>A-4-62</td>
			<td>Rotor</td>
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			<td>Fox Lifesciences</td>
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			<td>XF 100 mM Pyruvate Solution</td>
			<td>Agilent</td>
			<td>103578-100</td>
			<td>Reagent</td>
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		<tr>
			<td>XF 200 mM Glutamine Solution</td>
			<td>Agilent</td>
			<td>103579-100</td>
			<td>Reagent</td>
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		<tr>
			<td>XF DMEM medium</td>
			<td>Agilent</td>
			<td>103575-100</td>
			<td>Reagent</td>
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		<tr>
			<td>XFe96 FluxPak</td>
			<td>Agilent</td>
			<td>102601-100</td>
			<td>Material</td>
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		<tr>
			<td>Xylazine (AnaSed Injection)</td>
			<td>DAILYMED</td>
			<td>NDC 59399-110-20</td>
			<td>Anesthetic</td>
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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...

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Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

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 ...

This protocol focuses on quantifying mitochondrial respiration of mouse and human neutrophils, as well as neutrophil-like HL60 cells using the metabolic extracellular flux analyzer. This method allows us to quantify mitochondrial function in neutrophils under normal and disease conditions. This is a straightforward technique that can deliver insights into the mitochondrial function of the cells in response to drugs or any disease condition.Since different cells have different mitochondrial respiratory efficiency, optimization of cell seeding density and reagent concentration has to be performed accurately to get high-quality results. To begin the metabolic extracellular flux assay, take a sensor cartridge packed over a specially designed 96-well plate and transfer 200 microliters of calibration medium into each well of the underlying plate. Carefully lift the cartridge and the plate with calibrant, and place it in a non-carbon-dioxide humidified 37 degrees Celsius incubator overnight for hydration.Next, based on the cell type, coat the 96-well plate with a specific coating to ensure cell adhesion. Afterward, wash the plates with 200 microliters of sterile PBS twice. Prepare the assay medium in DMEM with one millimolar pyruvate, 10 millimolar glucose, and two millimolar glutamine.Procure the mouse and human neutrophil cells as described in the text manuscript. For the neutrophils procured from the mouse, adjust the cell density to 1.1 times 10 to the sixth cells per milliliter by adding assay medium. Then seed 180 microliters of the mouse neutrophil suspension per well into the prepared 96-well plate.Similarly, adjust the human neutrophil cell density to 2.2 times 10 to the sixth cells per milliliter using the assay medium. On the day of the assay, count the neutrophil-like HL60 cells manually using the hemocytometer. Then centrifuge the differentiated or undifferentiated HL60 cells at 300G at room temperature for five minutes.Wash the cells with DMEM once and re-suspend them in the assay medium to achieve a cell density of 1.39 times 10 to the sixth cells per milliliter. Next seed 180 microliters of differentiated or undifferentiated HL60 cells into the pre-prepared 96-well plate containing the mouse and human neutrophil samples. Finally, add complete medium to the corner wells of the cell culture plate devoid of cells for background correction while seeding.Centrifuge the plate at 300G at room temperature for three minutes without break to ensure proper adherence of the cells on the bottom of the plate. Finally, incubate the plate for one hour to pre-equilibrate the cells with the assay medium. Open the mitochondrial stress test kit and prepare the reagents as per the manufacturer's instructions.Next, load the cartridge with reagents, starting with the prepared oligomycin into port A, FCCP into port B, and rotenone/antimycin A mixture into Port C for injections. Fill the unused ports with 20 microliters of assay medium. Equilibrate the assay system at 37 degrees Celsius at least five hours before the assay by opening the Wave software and clicking the Heater On tab.After reaching the temperature, the lower left corner of the Wave software shows ready. Open a template for the mitochondrial stress assay kit in the software. Select injection strategies on the left side, then click Add and name the injection condition as human neutrophils or mouse neutrophils or HL60.Select port A to D by clicking on each, and click on Add Compound and enter oligomycin or FCCP or rotenone/antimycin A with the respective concentrations. Select Pretreatment on the left side, then click Add and name them CD18 and Cell Tak separately. Select the Cell Type on the left side.Then click Add and name them human neutrophils, mouse neutrophils, and HL60 or DHL60 with a feeding density as applicable. Define the groups by clicking on Add Group. Then click on the underlined definition.Click on Plate Map in the top menu to observe all the groups with definitions on the left side and the plate map on the right. Drag and drop to add each well to the group while maintaining the four corner wells as background. To set up the protocol, select the Protocol tab in the top menu, and define three cycles of baseline, oligomycin, FCCP, rotenone and antimycin A mixture, by setting the time to mix as three minutes, rest as zero minutes, and measurement as seven minutes.Go to the Run Assay page, provide the project summary information for reference, and click Start Run. Give the location to save the files so that all the results are saved after completion of the assay. Then click on Start Run again.After the automatic loading of the assay, wait for the tray to open to place the sensor cartridge and plate with 200 microliters of calibrant. Set the cartridge barcode direction facing to the right. Run the calibration by clicking I'm Ready, which takes approximately 20 minutes.After the calibration, click on Open Tray, replace the plate with the cell seeded plate, and click on I'm Ready to continue the assay. After completing the assay, the data is automatically saved. Export the results to a spreadsheet or other analysis software file.Plot the graph and analyze the data. The representative dynamics of oxygen consumption rate or OCR indicated changes in the mitochondrial respiration in mouse and human neutrophils, as well as undifferentiated and differentiated HL60 cells in response to oligomycin, FCCP, and the mixture of rotenone and antimycin A.The OCR values decreased in all cells after oligomycin treatment due to the inhibition of the proton channel of ATP synthase. FCCP treatment increased the flow of electrons and oxygen consumption to restore the OCR value and achieve maximal respiration.A mixture of rotenone and antimycin A blocked complexes 1 and 3 of the electron transport chain, leading to the elimination of mitochondrial respiration. Decreased mitochondrial respiration was observed in HL60 cells after neutrophil-directed differentiation. Differentiated HL60 cells were found to have significantly lower basal mitochondrial respiration, proton-leak-linked respiration, ATP-linked respiration, and non-mitochondrial respiration.The increase in maximal respiration of differentiated HL60 cells was not substantial, while the spare respiratory capacity was significantly increased. Make sure the cells are at the bottom of the plate, as the sensor cartridge should not have any obstacles due to the floating cells while reading the OCR value. This technique would help researchers to study the effect of drugs or mitochondrial dysfunction in neutrophil-associated studies.

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Abstract