Name: real-time measurement of the mitochondrial bioenergetic profile of neutrophils
Uploaded: 2023-06-02T13:00:00
Description: Watch this Scientific Journal Video about Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils at JoVE.com

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>37 &#176;C non-CO2&#160;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&#160;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

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...

Watch this Scientific Journal Video about Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils at JoVE.com

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 ...

Research

JoVE Journal

Immunology and Infection

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...