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 optimize the concentration42. It is also better to perform sequential additions of FCCP during the optimization. Although the metabolic extracellular flux analyzer injects the drugs into the plate under air pressure43, the impermeability of the cell membrane to some mitochondrial complex inhibitors limits their use in the case of intact cells in comparison to isolated mitochondria44. The loading into the ports has to be carefully performed to avoid cross-contamination. The substrate concentration (e.g., glucose, pyruvate, glutamine) in the medium may also influence mitochondrial activity and needs to be optimized.
Apart from the drug concentrations, the cell seeding density is also a critical parameter in obtaining a good OCR value. Optimal cell seeding density varies by cell type, and it is recommended to use different seeding densities in preliminary experiments to test the efficacy of the measurement. Accurate cell counting is mandatory for lowering the variability between groups, and the appropriate coating materials for the culture plate must be tested to ensure the adherence of cells at the bottom of the plate. Centrifugation of the culture plate with a speed of 300 &#215; g at RT for 5 min without brake helps the quick sedimentation and attachment of the cells. Before seeding the cells, sufficient washing is desirable after aspirating the coating material.
The seeding of cells is performed by pipetting cells at the bottom edge, to avoid edge effects and to ensure uniform spreading of the cells45. The cells are maintained at 37 &#176;C and allowed to rest for at least 1 hr to minimize the edge effect46. Maintaining them in a non-CO2 incubator to degas the cell culture plate before measurement is recommended since the level of oxygen in the gas mixture can differ according to the experiment and cell type, with different hypoxia values for different tissues and cell types47. The assay medium must be prepared on the day of the assay (70 mL of medium is sufficient for the assay). Normalization of the data with the cell number can be performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazolium bromide (MTT) assay, the nuclear fragmentation assay with Hoechst, the cell counting assay, or protein or DNA quantification assays48. Since different mitochondrial inhibitors are used, cell viability needs to be considered as a factor for the result. It is recommended to perform a cell viability test at the end of the assay to rule out a possible cytotoxic effect of the drugs used.
In addition to OCR, the metabolic extracellular flux analyzer can also measure ECAR, which is commonly used in quantifying glycolysis49. However, in the mitochondrial stress assay, ECAR values reflect the acidity generated by both glycolysis and the tricarboxylic acid cycle (via CO2) in mitochondrial respiration. To measure glycolysis, a glycolysis stress test kit is recommended. Initially, the OCR prior to the addition of the compounds shows the basal respiration of the neutrophils. Basal respiration occurs due to the protons transported from the mitochondrial matrix to the intermembrane space passing through the inner mitochondrial membrane, depending on the inner mitochondrial membrane composition. Basal respiration accounts for ~30%-50% of the metabolic rate of resting cells1,50.
Calculations based on the basal respiration of the assay could reduce the variability among groups and types of cells. Normalization of the data has been achieved by comparison to the basal respiration without the addition of any drugs, which allows normalizing changes depending on cell count and bench error on the assay. Spare respiratory capacity is the difference between maximal OCR and basal OCR and is an indicator of how close the cells are functioning to their bioenergetic limit. A decrease in maximal respiration indicates reduced electron transport in the respiratory chain from complexes I and II to complexes III and IV, and eventually to molecular oxygen. Figure 4A shows the schematic representation of the calculations of the assay.
The increase in spare respiratory capacity indicates that cells can respond well to further energy demands, which is a prerequisite in the case of neutrophils as they end up in respiratory burst on activation51,52,53,54,55. However, ECAR data of the mitochondrial stress assay cannot represent the glycolysis rate of the neutrophils. Certain assessments can be made on the data; for example, the reduction in ECAR values in both mouse and human neutrophils after the addition of rotenone and antimycin A implies that the ECAR values before the addition of these complex I and III inhibitors are primarily due to CO2 production from the TCA cycle. In the case of cell lines, the ECAR values are constant after the addition of rotenone and antimycin A, suggesting the ECAR values before the addition are due to glycolysis45.
Mitochondrial disorders are clinical conditions characterized by faulty OXPHOS. In the case of neutrophils, measurement of the OCR is low due to no cell proliferation, low mitochondrial count, and early senescence56. In neurodegenerative disorders, neutrophils extravasate into amyloid-&#946; (A&#946;) deposit areas. The A&#946;42 peptide triggers integrin activation and rapid neutrophil adhesion57. During apoptosis, neutrophil mitochondria release proapoptotic proteins into the cytosol58. It is also shown that the neutrophil migration speed is reduced, and chemotaxis is abolished when treated with CCCP9. This suggests the potential role of mitochondria and mitochondrial respiration in neutrophil function. Mitochondrial activities in diverse cell types under many pathological conditions have been reported and linked to pathogenesis, such as Alzheimers5, schizophrenia59, chronic respiratory diseases60, bipolar disorders, sepsis, diabetics, asthma, pulmonary hypertension, and sickle cell disease. Emerging therapies, such as improving respiratory chain flux by using antioxidants (e.g., CoQ10, idebenone, alpha-lipoic acid, vitamin C and E), and/or cofactors (e.g., riboflavin, thiamine), and the administration of mitochondrial substrates such as L-carnitine, have been used in treating mitochondrial disorders. However, these treatments are not standardized and may not be effective61. A noninvasive standardized methodology, such as using the metabolic extracellular flux analyzer, to quantify mitochondrial functions in disease-relevant cells, such as neutrophils, could serve as a diagnostic biomarker in therapeutics.

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

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 increasing the flow of electrons and oxygen consumption to maintain the membrane potential and achieve maximal respiration; and rotenone/antimycin A mixture treatment eliminates mitochondrial respiration by blocking complexes I and III of the electron transport chain.
We observed that after neutrophil-directed differentiation, HL60 cells showed decreased mitochondrial respiration (Figure 3C). After quantifying different respiration parameters, mentioned above, differentiated HL60 cells showed significantly lower basal mitochondrial (Figure 4B) respiration, proton leak-linked respiration (Figure 4C), ATP-linked respiration (Figure 4D), and non-mitochondrial respiration (Figure 4G). Maximal respiration (Figure 4E) in differentiated HL60 cells was increased, but not significantly. The spare respiratory capacity (Figure 4F) was significantly increased.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64971/64971fig03.jpg" /> 
Figure 3: Representative graphs showing the dynamic changes of OCR during the mitochondrial stress test assay. (A) Mean &#177; SD from n = 3 replicates of mouse neutrophils. (B) Mean &#177; SD from n = 3 replicates of human neutrophils. (C) Mean &#177; SD from n = 3 replicates of undifferentiated (HL60, blue) and differentiated (dHL60, red) HL60 cells. Abbreviation: OCR = oxygen consumption rate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64971/64971fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64971/64971fig04.jpg" /> 
Figure 4: Respiration parameters obtained from OCR dynamics. (A) Schematic showing how to calculate (a) basal mitochondrial respiration, (b) proton leak-linked respiration, (c) ATP-linked respiration, (d) maximal respiration, (e) spare respiratory capacity, and (f) non-mitochondrial respiration. (B-G) Mean &#177; SD of n = 3; (B) basal mitochondrial respiration, (C) proton leak-linked respiration, (D) ATP-linked respiration, (E) maximal respiration, (F) spare respiratory capacity, and (G) non-mitochondrial respiration of undifferentiated (HL60) and differentiated (dHL60) HL60 cells. ns (non-significant) p &#62; 0.05, *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001 by unpaired Student&#39;s t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64971/64971fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

real-time-measurement-mitochondrial-bioenergetic-profile

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.8K Views.  UConn Health. We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Video: Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

Cancer Research

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 neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

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

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Biology

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is ...

Measuring Mitochondrial Function of Na&#239;ve and Effector CD8 T Cells

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. Lymphocyte biology has been explored using bioenergetic analysis and has now become a critically import tool in the field. Thus, we sought to optimize a bioenergetic analysis assay that can be adapted with pretreatments and acute injection for receptor stimulations. Here, we evaluated CD8 T cell ex vivo metabolism using the Cell Mito Stress Test to assess rates of oxygen consumption and extracellular acidification in na&#239;ve and effector CD8 T cells. Antigen-specific effector CD8 T cells were derived via ex vivo stimulation, and na&#239;ve CD8 T cells harvested from splenocytes and isolated with magnetic bead column separation. 
Pretreatments are performed in microplates and we detail how to prepare sensor cartridges. We show how injection ports can loaded with drugs to indirectly measure metabolic capacities and with metabolic modulators, this protocol can be used to study specific enzyme activity. T-cell receptor stimulations can be studied in real time with acute injection and stimulation with anti-CD3/CD28 using the injection ports. Instrument analyzers are used for measurements and data collection and data visualization is done with software programs to interpret cellular metabolism. This strategy produces an extensive amount of data on immune cell biology and mitochondrial bioenergetics allowing researchers to customize the protocol in numerous ways to explore CD8 T cell metabolism.

CD8 T cell bioenergetics can be interrogated using the Mito Stress Test. This methodology can be used to study acute and chronic metabolic programming. This protocol describes approaches to examine the relationships between T cell receptor biology and bioenergetic analysis.

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. ...

MATLAB Optimization for Analyzing Mitochondrial Networks in Neuroscience

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

Author Spotlight: An Optimized Automated Method for Investigating Retinoic Acid Receptors in Neuronal Mitochondria

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...

Neuroscience

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring &#916;&#936;m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting &#916;&#936;m fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study &#916;&#936;m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these ...

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.

Author Spotlight: Unveiling Mitochondrial Function and Cellular Metabolic Adaptation in Metabolic Diseases

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...