This work was supported by funding from the Academy of Finland (C.B.J), the Magnus Ehrnroot Foundation (C.B.J), and a Doctoral fellowship of the Integrated Life Sciences Graduate School (R.A.).

No conflict of interest to disclose.

Traditionally, mitochondrial bioenergetics&#160;has been studied with Clark-type oxygen electrodes. A lack of resolution and throughput, however, warranted for technological advancement. To date, the O2k (referred to as cHRR) and Seahorse XF96 Flux Analyzer (referred to as mHRR) have been widely adopted in the field of cellular bioenergetics. Here, we present a comprehensible collection of protocols for the analysis of cellular energy metabolism via assessment of mitochondrial respiration using either cHRR or mHRR, discuss key benefits of each device and provide practical guidance. The protocols provided here encompass mammalian cells, fibrous (hard) tissues such as skeletal and heart muscle, and non-fibrous (soft) tissues such as brain and liver and are applicable to similar types of sample material.
While both HRR methods result in comparable data for mammalian cells as exemplified with HEK293 with multiple OXPHOS deficiency, general working principles and technical setup of the devices render them suitable for different applications. The mHRR setup allows automated data acquisition and has high-throughput capability with a 24- or 96 multi-well setup requiring minimal sample amounts. However, the low experimental volume and well surface, in addition to the use of oxygen-permeable polymers (polystyrene), can cause high intra-well variation (especially in the 96-well plate setup), promoting the use of &#8805;6 wells per condition for reproducible results. In contrast to the cHRR based-polarographic oxygen sensor, no oxygen is consumed by the sensor probes of the mHRR, which utilizes quenched phosphorescence O2 sensing. As the mHRR is a semi-closed system, ambient O2 can diffuse into the respiration medium, exposing the sample and the probe to oxygen. When the piston-like sensor probe lowers, a temporarily sealed and isolated chamber is created to amplify changes in O2 concentration and measure oxygen consumption. Subsequently, a mathematic model is employed to accurately compute oxygen consumption rates by estimating the back diffusion of O2. However, the drawback is that the algorithm also amplifies noise29. The microplate setup utilizes non-reusable specialized ports and plates requiring optimal cell seeding density. The mHRR oxygen sensor probes measure lateral O2 diffusion in three distinct areas per well equivalent to the size of the probe (~1 mm); therefore, a uniformly distributed cell monolayer is crucial to determine accurate oxygen consumption rates. If any significant gaps are present when observing the cell distribution, incorrect oxygen consumption rates will be computed, resulting in a high variance of well replicates. Taking this into consideration, mHRR is semi-automated and ideally adaptable for high-throughput cell or small-organism-based studies (e.g., C. elegans) with recurring screenings.
The cHRR respirometers setup is based on a two-chamber system with polarimetric measurement of oxygen. In the closed-based system, ambient O2 cannot diffuse into the respiration medium; thus, a decline in O2 concentration reflects oxygen consumption of the biological sample. As the cHRR polarographic oxygen sensor consumes oxygen, diffusion of free O2 is essential, making it difficult to minimize chamber volume (2 mL, new cHRR devices 0.5 mL) and requiring constant stirring to ensure homogeneity of the respiration medium. Consequently, larger sample volumes are required to generate sufficient oxygen flux. Due to direct access to the chambers and manual titration, any respiration protocol is adaptable and based on widely commercially available chemicals results in exceptionally low running costs. In addition, ad hoc operability allows adapting a SUIT protocol by titration during an experiment and is important in single-time patient-derived samples. Partial automation is feasible using a titration-injection micropump, which enables programmable SUIT protocols. Although restricted to two samples per cHRR device, experienced users will run several devices in parallel. The benefit of the versatility of assay development and software environment comes with the need for more specific training to operate these devices and user-dependent maintenance (e.g., calibration of polarographic oxygen sensors) in order to acquire reproducible data. For advanced applications, additional modules are attachable to the cHRR devices to record pH, fluorescent module to assay membrane potential via safranin30, H2O2 via AMPLEX red24, and calcium levels31.
Both devices require specialized respiration media. mHRR uses a commercially available serum-free growth medium, avoiding the use of bicarbonate, which degasses under low CO2 conditions and is essential if extracellular acidification rate (ECAR) is of importance. During glycolysis, pyruvate is converted into lactate, which dissociates into lactic acid and protons. This increased proton concentration causes the pH to decrease, which is recorded as ECAR. A more elaborate analysis of ECAR is possible with the glycolysis stress test. This assay consists first of adding saturating glucose levels to measure the basal glycolytic rate. After inhibition of the ATP synthase complex with oligomycin, the maximal glycolytic rate is revealed. The final step is to measure non-glycolytic acidification by injecting 2-deoxy-glucose, which inhibits glycolysis via competitive binding to hexokinase and phosphoglucose isomerase32. Other kit-based protocols available encompass glycolysis, fatty acid oxidation, and glutamine oxidation. Here, we used the standard protocol to measure basal respiration, ATP-linked respiration, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration (Figure 4A-C). An approach of utilizing both the standard respiration protocol in conjunction with the glycolysis stress test would give insight into aerobic and anaerobic energy pathways providing an overview of cellular respiration.
Cellular bioenergetics is usually accessed in adherent cells with the microplate mHRR setup. Dependent on cell type, specialized coating for adherence (e.g., poly-D-lysine, gelatin, etc.) might be required (for suspension cells) as well as for loosely adherent cells as they may detach from the bottom of the well measurement cycles33. In contrast, cHRR measurements require constant stirring, allowing respiratory measurement for any biological sample. For each device, optimization such as titration of inhibitors (and substrates) for tissue and cell lines to assess inhibitor-susceptibility (dose-response curves) is required. Inhibitor concentrations should be trialed in any experimental model to use the lowest fully inhibiting concentrations and to prevent unspecific inhibition as well as unnecessary chamber contamination. Generally, 0.25 &#181;M rotenone, 0.5 &#181;g / mL oligomycin, 0.5 &#181;g / mL antimycin are sufficient for most applications and sample amounts. For reproducibility, prepare single-use drugs in sufficient quantities for the entire planned experiments (e.g., mouse groups) and keep sample input constant within a specific tissue or cell line. In cHRR, traces of inhibitors remaining in the chambers will alter flux without any indication to the operator. Particularly rotenone traces are difficult to evade and require sufficient washing with a minimum chamber (and stopper) cleaning, which encompasses washing 4x with ddH2O, 2x 70% EtOH (96% purity sufficient), 1x 100% EtOH, 1x 70% EtOH. Washing requires turning stirrers and keeping EtOH-steps for &#62; 5 minutes each. To prevent bacterial contamination, 70% EtOH is kept in all chambers when machines are not in use. Potential residual contaminates can be quenched by the addition of unused tissue lysate. In a mHRR experiment, certain chemicals may interact with the essential single-use plasticware34.
Respiratory data obtained via a permeabilized protocol give insight into the ETS, whereas an intact protocol provides insight into mitochondrial properties such mitochondrial coupling efficiency. Indeed, general mitochondrial energy properties can give the same outcome, but underlying electron transfer between complexes may be altered. This would reflect altered mitochondrial energy metabolism and requires a permeabilization protocol to access the respiratory complexes. Similarly, different tissues and cells show altered substrate dependency when assessing respiratory characteristics. For instance, glycolytic muscle fibers are known to rely primarily on glycerol-3-phosphate for energy delivery, whereas oxidative muscle fibers possess a two-fold higher electron transfer capacity from NADH oxidation35,36. On the same note, heart mitochondria, liver, brown adipose tissue can utilize fatty acids to synthesize ATP, and in turn, the brain can use ketone bodies, predominantly formed by fatty acid oxidation36,37,38. Therefore, determining mitochondrial characteristics requires a holistic approach as opposed to the standard glucose-dependent respiration. mHRR usually encompasses assessing intact adherent cells, although permeabilization is achievable (e.g., mutant recombinant perfringolysin O that selectively permeabilizes the cell membrane39). However, these methods come with increased complexity due to the limitation of four injection ports. In contrast, correctly lysed non-fibrous tissue lysates and any cell type are usually problem-free for cHRR. Normally, assaying tissue with the mHRR is not feasible; however, approaches using isolated mitochondria from a tissue have been established40,41.
Important to note is that normalization to whole protein content neglects absolute mitochondrial amounts. Comparison of experimental groups under various treatments can differ significantly (e.g., 30 days high-protein diet-fed rats showed a 2.5-fold increase in mitochondrial content in liver42), particularly in tissue susceptible to intracellular lipid accumulation such as brown adipose tissue, liver, and skeletal muscle. In these situations, it is also recommended to assess several mitochondrial markers as an approximation for mitochondrial mass, such as mtDNA copy number43, citrate synthase activity10, and ubiquitous mitochondrial proteins (e.g., VDAC1, TOM20). In combination, this allows to distillate whether an altered respiratory function is attributed to mitochondrial quantity, quality, integrity, or a combination thereof. Another complementary method to this is the implementation of FCRs. FCRs give insight into different respiratory states independent of mitochondrial content. FCRADP derives whether altered LEAK or OXPHOS change the efficiency of the mitochondria to phosphorylate ADP. FCRstate 3 (I) reflects&#160;to what degree the sample is dependent on complex I as a comparison to complex II. FCRstate 3 (II) compares succinate-dependent respiration with ETS and provides an index for mitochondrial respiration derived from complex II. FCRcoupled/uncoupled is a ratio providing the coupling control between OXPHOS and ETS, with a ratio of 1 having no spare respiratory capacity left. The mitochondrial outer membrane integrity can be assessed through the addition of exogenous cytochrome c. Cytochrome c is localized in the intermembrane space, where it facilitates the transfer of electrons between complex III and complex IV. If the outer mitochondrial membrane is damaged, cytochrome c leaks out of the mitochondria, not contributing to respiration anymore. Restoring this imbalance can be achieved via the addition of exogenous cytochrome c, consequently increasing respiration (Figure 1A, D; 5B). Complex IV activity is measured individually with TMPD after complete inhibition of OXPHOS. Baseline O2 flux will decline as the O2 concentration falls because TMPD oxidation is O2 concentration-dependent. Hence after ROX assessment, all chambers are oxygenated to equal oxygen level (150 &#181;M). Linear regression of data points from signal after addition of azide can be used to interpolate chemical and O2-dependent background of complex IV activity assay. ROX values should be subtracted from all respiration values to correct for non-mitochondrial oxygen consumption.
Biologically, ROX in the case of isolated mitochondria can be lower than with permeabilized or intact cells/tissue. In general, residual respiration is caused by the activity of oxidase enzymes, with cells and tissue having more autoxidizable substances than isolated mitochondria44. Furthermore, with intracellular membrane structures still intact, the difficulty of oxygen to permeate through cell membranes increases due to its negative charge. Consequently, diffusion through cellular membranes and intracellular availability of oxygen can be affected, resulting in ROX. However, isolation of mitochondria has been shown to disrupt mitochondrial morphology, increase mitochondrial hydrogen peroxide production, along with altered mitochondrial respiratory function45. While isolation of functional mitochondria allows for specific normalization and might be required in certain conditions, the isolation process is time-consuming, usually requires more material, and might not preserve mitochondrial heterogeneity. For this reason, we have refrained from including mitochondrial isolation in this study. However, for skeletal or heart muscle, isolation of mitochondria or saponin-treatment of fibers is essential for respiration measurements. As autolytic processes occur very quickly after euthanasia, fast tissue extraction and adhering to comparable timing for preparations between individual experiments is recommended.
In our experiments, assessment of the mitochondrial respiratory function of mammalian cells led to comparable results with both HRR devices. However, basal respiration was higher for cHRR compared to mHRR. Apart from numerous technical aspects (chamber volume, stirring and differing signal integration), biological reasons such as altered respiration medium, timing of cell harvesting and non-attached cells causing loss of cell contact could inflict the observed differences. Consequently, respiration protocols in &#160;general and individual substrate and inhibitor concentrations are not interchangeable between systems for the presented reasons, which could be technical in nature (e.g., titration) and generally differing assay reagents (e.g., respiration media, chemical absorbances of glass or polymers). To ensure highest reproducibility, several technical considerations and recommendations encompass (i) the use of single-use aliquots to minimize cross-contamination or freeze-thaw cycles, (ii) appropriate storage of all chemicals (e.g., ADP at -80 &#176;C for prolonged stability, pyruvate prepared freshly and light-sensitive chemicals in the dark), (iii) regular and rigorous re-testing of chemicals for efficacy (e.g., evaporation-inflicted concentration changes, storage-induced TMPD activity loss) and (iv) extensive cleaning to remove any trace chemical. Considerations on the reproducibility of longitudinal studies (over several years) would require monitoring the device performance and ensuring the stability of reagents over time.
Finally, novel technology based on combined potentiometric (pH) and amperometric (O2) measurements through ruthenium oxide-based electrodes could catalyze a paradigm shift in current tools, allowing studying cellular metabolism in culture and in vivo46. Although current methods allow predominantly ex vivo and in vitro assessment of cellular metabolism, delayed fluorescence enables in vivo analysis of mitochondrial oxygen as a measure of mitochondrial function47. Similarly, microfluidics-based respirometry shows promise in higher sensitivity, requiring only a few hundred cells48. While new methodologies are on the horizon, to date, high-resolution respirometry remains the gold standard to assess cellular respiration capacity for which quintessential protocols are provided here, applicable to most cells, tissues, and organisms to study mitochondrial respiration.

Mitochondria fulfill the key provision of energy and are a compartmentalized organelle contributing to essential cellular bioenergetic and metabolic processes such as anabolism of nucleotides, lipids and amino acids, iron-sulfur cluster biogenesis and are implicated in signaling such as controlled cell death1,2,3. Mitochondrial bioenergetics through oxidative phosphorylation contributes to almost all cellular processes within the cell, and consequently, mitochondrial dysfunctions of primary or secondary origin are associated with a wide spectrum of disease conditions4,5. Mitochondrial dysfunction not only involves alterations in structure or mitochondrial density but also in the quality and regulation of the respiratory system6. This qualitative element encompasses substrate control, coupling characteristics, post-translational modifications, cristae dynamics, and respiratory supercomplexes7,8. Therefore, accurate analysis of mitochondrial bioenergetics for experimental and diagnostic approaches to assess the energy metabolism of the cell is important in health and disease.
Mitochondrial oxidative phosphorylation (OXPHOS) is a sequence of reactions within the respiratory system or electron transfer system (ETS) for the generation of cellular energy through adenosine triphosphate (ATP)9. The multi-enzymatic step to harness energy from electron flow through complexes I and II to complex IV generates an electrochemical proton gradient across the inner mitochondrial membrane, subsequently utilized for phosphorylation of adenosine diphosphate (ADP) to ATP via complex V (F1FO ATP synthase) (Figure 1A).
First, two-electron carriers are generated during the tricarboxylic cycle (TCA), glycolysis, and pyruvate oxidation: nicotinamide adenine dinucleotide (NADH) and dihydroflavine adenine dinucleotide (FADH2). NADH is oxidized at complex I (NADH dehydrogenase), during which two electrons are transferred to coenzyme Q (quinone is reduced to quinol), while protons are pumped into the intermembrane space (IMS). Second, complex II (Succinate dehydrogenase) oxidizes FADH2 and feeds the electrons to coenzyme Q without pumping protons. Third, at complex III (Cytochrome c oxidoreductase), electrons from coenzyme Q are transferred to cytochrome c while protons are pumped into the IMS. Fourth, cytochrome c transfers the electrons to complex IV (Cytochrome c oxidase), the final complex to pump protons, and where oxygen functions as an electron acceptor to assimilate protons, ultimately forming water. It is this oxygen that mitochondria consume which can be measured by an oxygraph. Finally, the protons generated from complex I, complex III, and complex IV are used to rotate complex V, thereby generating ATP9.
Importantly, electron transfer occurs not only in a linear fashion, otherwise denoted as the electron transport chain. Instead, electrons can be transferred to the coenzyme Q pool through multiple respiratory pathways and facilitate convergent electron flow. NADH-substrates and succinate, for example, can enter via complex I and complex II, respectively. Electrons from fatty acid oxidation can be donated via the electron transferring flavoprotein complex. Indeed, a comprehensive analysis of OXPHOS requires a holistic approach with appropriate fuel substrates (Figure 1A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63000/63000fig1v2.jpg" /> 
Figure 1: Mitochondrial oxidative phosphorylation and specific substrate and inhibitor protocols. (A) Mitochondrion and scheme of the electron transfer system (CI-CIV) and mitochondrial F1F0 ATP synthase (CV). All structures are from PDB. The figures only depict substrates and inhibitors described in this study). (B) Sample trace of oxygen flux in intact HEK293 cells using standard protocol in a mHRR device. (C) Sample trace of oxygen flux in intact HEK293 cells using standard protocol in a cHRR device. (D) Sample trace of oxygen flux in permeabilized human fibroblasts from a healthy donor with respective SUIT protocol. Abbreviations: 1 = Routine respiration of intact cells; 2 = State 2; 3 = State 3(I); 4 = State 3(I) with cytC; 5 = State 3 (I+II); 6 = Leak(OM); 7 = ETS capacity; 8 = S(ROT); 9 = ROX; 10 = TMPD; 11 = Az. ROT = Rotenone, AM = Antimycin, ATP = Adenosine triphosphate, Az = Azide, OM = Oligomycin, FCCP = Carbonyl cyanide p-trifluoro-methoxyphenyl-hydrazone; Asc = Ascorbate, TMPD = N,N,N&#8242;,N&#8242;-tetramethyl-p-phenylenediamine, Succ = Succinate, M = Malate, P = Pyruvate, ADP = Adenosine diphosphate, NAD = Nicotinamide adenine dinucleotide, IMS = Intermembrane space, FAD = Flavin adenine dinucleotide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63000/63000fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Analysis of mitochondrial OXPHOS capacity using HRR has become an instrumental biochemical method of diagnostic value not only for primary mitochondrial defects10,11 but extending to all other realms of biology such as cancer and ageing12. HRR allows the determination of cellular respiration by the analysis of mitochondrial OXPHOS capacity, which directly reflects individual or combined mitochondrial respiratory complex deficiency, and indirectly is associated with cellular dysfunction and altered energy metabolism9. Methodologically, respiration measurements are performed using cells, tissue, or isolated mitochondria11,13,14, with frozen material only partially suitable15,16. Frozen tissue is shown to have an intact ETS with maintained supercomplex stability15. Thus, as opposed to traditional TCA intermediates, respective substrates are directly fed into the ETS. However, coupling between the ETS and ATP synthesis is lost as the membrane integrity is compromised through freeze damage (ice crystal formation).
Respiration experiments normally take place at a physiological temperature of 37 &#176;C for endotherms in either non-permeabilized or permeabilized cells or tissue. While the former considers the cytosolic metabolic context, the latter provides the energetic contribution of individual OXPHOS complexes and the ATPase through the addition of specific substrates (and inhibitors). The sequence and variation of substrates and inhibitors have led to the development of a diverse array of SUIT protocols17 and assays18 to address various scientific questions of OXPHOS function (reviewed under12). The basic protocol of cellular respiration assesses four different states: i) routine respiration&#160;- the respiration in a respective respiration media without any addition of substrates or inhibitors consuming but endogenous substrates. This state can reveal general OXPHOS or secondary-induced respiration defects caused, for example, by altered metabolite profiles. Next, the addition of the ATPase inhibitor oligomycin reveals the permeability of the inner mitochondrial membrane to protons, defined as ii) leak respiration.&#160;Subsequent titration of a protonophore such as the uncoupler carbonyl cyanide p-trifluoro-methoxyphenyl-hydrazone (FCCP) allows to determine the state at which ETS capacity is maximal in an open-transmembrane proton circuit mode, defined as iii) uncoupled respiration. Importantly, an uncoupled state can also occur by experimental interventions through excessive mechanical damage to the mitochondrial membranes. Conversely, the non-coupled state refers to respiratory uncoupling by an intrinsic mechanism that is physiologically controlled. Finally, complete inhibition of the ETS by addition of the complex III inhibitor antimycin and complex I inhibitor rotenone determines residual oxygen consumption (ROX) from non-mitochondrial oxygen-consuming processes (Figure 1A-C).
Mitochondrial bioenergetics consists of five distinct respiration states19,20. State 1 respiration is without any additional substrates or ADP, except for what is endogenously available. After the addition of ADP, but still, no substrates, state 2 respiration is achieved. When substrates are added, allowing electron transfer and ATP synthesis, state 3 respiration is reached. In this state, OXPHOS capacity can be defined at saturating concentrations of ADP, inorganic phosphate, oxygen, NADH- and succinate-linked substrates. State 4 respiration or LEAK respiration can be defined as a state without ADP or chemically inhibited ATP synthases while having sufficient substrates. Lastly, when all oxygen is depleted (anoxic) in a closed-chamber setting, state 5 respiration is observed.
Several methods exist to assess cellular energy states14 with two devices dominating the current real-time assessment of OXPHOS through analysis of oxygen consumption, measured as the function of the decrease in oxygen over time in a closed-chamber system with different applicability dependent on the experimental model and research question: the Oroboros 2k high-resolution respirometer and the Seahorse XF extracellular flux analyzer. Both devices record the oxygen consumption rates as a decrease in picomoles (pmol) of oxygen (O2) per second as an absolute value within the chamber or microplate well. The specific oxygen consumption per mass is obtained by normalizing the respective oxygen consumption in a specific buffer recipe per number of cells (millions), tissue weight (mg), or protein amount.
The O2k (Oroboros Instruments) is a closed two-chamber system equipped with a polarographic oxygen sensor (abbreviated as chamber-based high-resolution respirometer: cHRR). Each experimental chamber holds 2 mL of liquid which is kept homogenous by magnetic stirrers. The polarographic oxygen sensor utilizes an amperometric approach to measure the oxygen: it contains a gold cathode, a silver/silver chloride anode, and in between a KCI solution creating an electrochemical cell upon which a voltage (0.8 V) is applied. Oxygen from the assay medium diffuses through a 25 &#181;m fluorinated ethylene propylene membrane (O2-permeable) and undergoes reduction at the cathode, producing hydrogen peroxide. At the anode, silver is oxidized by hydrogen peroxide, generating an electric current. This electric current (ampere) is linearly related to the partial oxygen pressure. The partial pressure of oxygen and the oxygen solubility factor of the assay medium are used to compute the oxygen concentration. Since oxygen partial pressure is dependent on experimental temperature and polarographic measurements are temperature-sensitive, fluctuations in temperature need precise (&#177;0.002 &#176;C) regulation by a Peltier heating block. Temperature can be controlled within a range of 4 &#176;C and 47 &#176;C.
The Seahorse XF extracellular flux analyzer (Agilent) is a plate-based system with 24- or 96-well microplate format in which three fluorescence electrodes measure oxygen consumption over time in each well (abbreviated as microplate-based high-resolution respirometer: mHRR). A maximum of four ports in the assay cartridge are available for automated injection during the assay. An assay contains multiple cycles, each with three phases: 1) mixing, 2) waiting, and 3) measurement. During the measurement phase, sensor probes are lowered into the microplate creating a temporarily closed chamber containing 7-10 &#181;L volume to measure emitted light. This light is emitted by polymer-embedded fluorophores on the tip of the sensor probes, which sense O2 based on phosphorescence quenching. The intensity of the fluorescence signal is proportional to O2 and influenced by the temperature of the sensor and assay medium. Therefore, accurate oxygen estimation requires a relative approach with a background well without any sample. Restoring oxygen concentration occurs during the mixing phase when the sensor moves up and down to mix the volume above the temporary chamber. Each cycle computes one oxygen consumption rate. Temperature can be controlled within a range of 16 &#176;C and 42 &#176;C.
HRR is the gold standard to assess cellular bioenergetics in primary and mitochondria-associated diseases and general cellular metabolism. In this study, basic protocols for HRR are provided to assess OXPHOS function in cells and tissues.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63000/63000fig2v2.jpg" /> 
Figure 2: Workflow for cell and tissue preparations for cHRR, and cell preparation for mHRR respirometry. (A) Outline of provided protocols. (B) Mammalian cells (step 1.2): HEK293 pellet equaling 3 x 106 cells (left panel). Non-fibrous tissue (step 1.3): Preparation of murine cerebellum lysate in 2 mL Teflon potter (middle panel). Saponin-induced skeletal muscle permeabilization (step 1.4) right panel) for cHRR respirometry. (C) Standard microplate seeding layout (step 2.4) and confluency check for the analysis of eukaryotic cells (HEK293) for mHRR respirometry. (D, E) Scheme of injection port loading for mHRR respirometry (step 2.4). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63000/63000fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>2 mL Potter-Elvehjem Glass/PTFE Tissue Grinder/Homogenizer</td><td>Omni International</td><td>07-358029</td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;, 5% CO&lt;sub&gt;2&lt;/sub&gt; medical gas mixture</td><td></td><td></td><td>Potter for tissue grinding</td></tr><tr><td>ADP</td><td>Sigma</td><td>A 4386</td><td></td></tr><tr><td>Antimycin A</td><td>Sigma</td><td>A 8674</td><td>Chemical</td></tr><tr><td>Ascorbate</td><td>Merck</td><td>PHR1279-1G</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>BSA (fatty accid free)</td><td>Sigma</td><td>A 6003</td><td>Chemical</td></tr><tr><td>CaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>C 4830</td><td>Chemical</td></tr><tr><td>Cytochrome c</td><td>Sigma</td><td>C 7752</td><td>Chemical</td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D 5628</td><td>Chemical</td></tr><tr><td>Dithiothreitol</td><td>Sigma</td><td>D 0632</td><td>Chemical, dissolve in DMSO</td></tr><tr><td>D-Sucrose</td><td>Roth</td><td>4621.1</td><td>Chemical</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (High glucose)</td><td>Fisher Scientific</td><td>41965-039</td><td>Chemical</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium&amp;nbsp;(No Glucose)</td><td>Fisher Scientific</td><td>A14430-01</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E 4378</td><td></td></tr><tr><td>Etomoxir</td><td>Sigma</td><td>E1905</td><td>Chemical</td></tr><tr><td>Falcon 15 ml Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>AM12500</td><td>Chemical</td></tr><tr><td>Falcon 50 ml Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>AM12501</td><td></td></tr><tr><td>FCCP</td><td>Sigma</td><td>C 2920</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7021</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Glutamate</td><td>Sigma</td><td>G 1626</td><td>Chemical</td></tr><tr><td>GlutaMax&amp;nbsp;(100x)&amp;nbsp;(200 nM&amp;nbsp;L-alanyl-L-glutamine dipeptide)</td><td>Fisher Scientific</td><td>35050061</td><td>Chemical</td></tr><tr><td>HEK293 cells</td><td>ATTC</td><td>CRL-1573</td><td></td></tr><tr><td>Hemocytometer</td><td>Fisher Scientific</td><td>0267151B</td><td>Instrument for cell counting</td></tr><tr><td>Hepes</td><td>Sigma</td><td>H 7523</td><td>Chemical</td></tr><tr><td>Imidazole</td><td>Fluka</td><td>56750</td><td>Chemical</td></tr><tr><td>KCl</td><td>Merck</td><td>1.04936</td><td>Chemical</td></tr><tr><td>L-carnitine</td><td>Sigma</td><td>C0283</td><td>Chemical</td></tr><tr><td>Malate</td><td>Sigma</td><td>M 1000</td><td>Chemical</td></tr><tr><td>MES hydrate</td><td>Sigma</td><td>M8250</td><td>Chemical</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M 9272</td><td>Chemical</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;ATP</td><td>Sigma</td><td>A 2383</td><td>Chemical</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;Phosphocreatine</td><td>Sigma</td><td>P 7936</td><td>Chemical</td></tr><tr><td>Na-pyruvate&amp;nbsp;(100 mM)&amp;nbsp;(100x)</td><td>Fisher Scientific</td><td>11360070</td><td></td></tr><tr><td>NEAA&amp;nbsp;(Non-essential amino acids) 100x</td><td>Fisher Scientific</td><td>11140035</td><td></td></tr><tr><td>Normal FBS&amp;nbsp;(10x)</td><td>Fisher Scientific</td><td>10500064</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;k-Core: Oxygraph-2k</td><td>Oroboros Instruments</td><td>10000-02</td><td>High-resolution respirometry instrument</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;k-Titration Set</td><td>Oroboros Instruments</td><td>20820-03</td><td>Hamilton syringes for chemical injections</td></tr><tr><td>Oligomycin</td><td>Sigma</td><td>O 4876</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Palmitoylcarnitine</td><td>Sigma</td><td>P 4509</td><td>Chemical</td></tr><tr><td>Penicillin-Streptomycin</td><td>Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma</td><td>P 2256</td><td>Chemical</td></tr><tr><td>RIPA-Buffer</td><td>Fisher Scientific</td><td>89900</td><td>Chemical</td></tr><tr><td>Rotenone</td><td>Sigma</td><td>R 8875</td><td>Chemical, dissolve in ethanol</td></tr><tr><td>Saponin</td><td>Sigma</td><td>S7900</td><td>Chemical</td></tr><tr><td>&lt;br/&gt; Seahorse XF DMEM assay medium pack, pH 7.4</td><td>&lt;br/&gt; Agilent, Santa Clara, CA</td><td>103680-100</td><td></td></tr><tr><td>Seahorse XFe96 Extracellular Flux Analyzer</td><td>&lt;br/&gt; Agilent, Santa Clara, CA</td><td></td><td>High-throughput respirometry instrument</td></tr><tr><td>Seahorse XFe96 FluxPak</td><td>&lt;br/&gt; Agilent, Santa Clara, CA</td><td></td><td>&lt;br/&gt; Includes assay plates, cartridges, loading guides for transferring compounds to the assay cartridge, and calibrant solution.</td></tr><tr><td>Small scissors</td><td>Fisher Scientific</td><td>08-951-20</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>Chemical</td></tr><tr><td>Succinate</td><td>Sigma</td><td>S 2378</td><td>Chemical</td></tr><tr><td>Taurine</td><td>Sigma</td><td>T 8691</td><td>Chemical</td></tr><tr><td>TMPD</td><td>Sigma</td><td>T 3134</td><td>Chemical</td></tr><tr><td>Trypan Blue solution</td><td>Merck</td><td>72-57-1</td><td>Chemical</td></tr><tr><td>Trypsin 0.25% EDTA</td><td>Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Two thin-edged forceps</td><td>Fisher Scientific</td><td>12-000-122</td><td></td></tr><tr><td>Uridine stock (500x)</td><td>Sigma</td><td>U3750</td><td>Chemical</td></tr></tbody></table>

high-resolution respirometry to assess bioenergetics in cells and tissues using chamber- and plate-based respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Here, we provide protocols to determine the mitochondrial bioenergetics in eukaryotic cells, non-fibrous tissue (e.g., cerebellum), and fibrous tissue (e.g., skeletal muscle). For eukaryotic cells, HEK293 with CRISPR-engineered knockout of two different proteins associated with mitochondrial translation resulting in multiple (CRISPRKO1) and severe/complete OXPHOS deficiency (CRISPRKO2) were measured with either cHRR (Figure 3A-C) or mHRR (Figure 3A-D).
For cHRR, HEK293 cells were digitonin-permeabilized, and respiration experiments performed following the standard protocol (step 1.5-1.6) and were successfully recorded (Figure 3A). CRISPRKO1 shows impaired and CRISPRKO2 no respiration compared to WT when normalized to cell input amount (Figure 3B). Protein amount was determined from collected samples (section 3), and values of routine respiration were normalized to protein amount in order to calculate absolute values and respective FCRs (Figure 3C, the meaning of each FCR is detailed in discussion). Optimal sample amounts produce fluxes of 80-160 pmol/s per mL. Ideally, the amount of cells or tissue is sufficient to generate a significant flux (20 pmol/s for cHRR) to reduce background noise while evading excessive reoxygenation during an experiment. In low respiring (e.g., white adipose fat, white blood cells) or hard-to-obtain samples (e.g., iPS-differentiated neuronal lineages), fluxes of 20 pmol/s per mL are sufficient in ideal working conditions.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63000/63000fig3v3.jpg" /> 
Figure 3: Representative standard protocol oxygen consumption traces from cHRR using HEK293 cells with combined OXPHOS deficiency. (A) Raw oxygen consumption traces of WT HEK293 cells and HEK293 cells with CRISPR-mediated mitochondrial translation defects causing multiple OXPHOS deficiency (CRISPRKO1,2). (B) Overlaid cell-input-normalized oxygen consumption traces from (A). (C) Protein-normalized quantification of two independent experiments (mean and SD) and respective FCRs. Comparisons between conditions were done by ANOVA and a posteriori Tukey&#39;s test or a Student&#39;s t-test. Significances: **** p &#60; 0.0001; *** p &#60; 0.001; ** p &#60; 0.01; * p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63000/63000fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Next, we used mHRR with the same cells in a standard protocol (2.1-2.5), confirming the OXPHOS deficiencies (Figure 4A). In addition, ECAR values were increased for severe/complete OXPHOS deficiency (CRISPRKO2), suggesting compensation of mitochondrial oxidative phosphorylation deficiency in HEK293 cells with specific mitochondrial translation deficiency through increased glycolysis resulting in lactate production (Figure 4A). Protein amount was determined from the microwell plate (section 3), and values obtained were normalized to protein amount (Figure 4B) and quantified (Figure 4C). Microplate-based systems are notorious for high intra-well variation. High variability between replicates can occur when the optimal seeding density has not been achieved; cells get detached during the washing steps of replacing the cell culture medium with assay medium, or improper pipetting technique such as the introduction of air bubbles or aspiration of varying volumes. Extended measurement times (6 measurement cycles) are recommended with mHRR to allow for stabilization of flux in media (Figure 1B and Figure 4A). Low fluxes cause high variation, and dependent on cell type, flux might be too close to background noise (up to 10-15 pmol/s). Low-respiring (e.g., fibroblasts) or exceptionally large cells might produce insufficient oxygen flux above background noise level in the 96-well microplate format even at 90% confluency. In that case, the 24-well mHRR or cHRR should be considered. Minimal changes in oxygen flux can also indicate faulty handling of loading the inhibitors, such as empty, incorrectly, or variably filled ports. The use of specific pipette tips that enter ports sufficiently during loading of the chemicals is recommended to allow chemicals to reach the individual port (Figure 2E). 
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63000/63000fig4v2.jpg" /> 
Figure 4: Representative standard protocol oxygen consumption traces from mHRR using HEK293 cells with combined OXPHOS deficiency. (A) Raw oxygen consumption traces of WT HEK293 cells and HEK293 cells with CRISPR-mediated mitochondrial translation defects causing multiple OXPHOS deficiency (CRISPRKO1,2). (B) Respective extracellular acidification rates (ECAR) from (A). (C) Protein-normalized oxygen consumption traces of WT HEK293 cells and HEK293 cells with CRISPR-mediated mitochondrial translation deficiency causing multiple OXPHOS deficiency (CRISPRKO1,2). (D) Protein-normalized quantification of wells (n = 8 per genotype; mean and SD). Comparisons between conditions were done by ANOVA and a posteriori Tukey&#39;s test. Significances: **** p &#60; 0.0001; *** p &#60; 0.001; ** p &#60; 0.01; * p &#60; 0.05. Abbreviations: see Figure 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63000/63000fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
An example experiment for non-fibrous tissue preparation (step 1.3 and 1.5-1.6) using mouse cerebellum (Figure 5A) and fibrous tissue preparation (step 1.4 and 1.5-1.6) using mouse skeletal muscle (soleus) is shown (Figure 5B). In general, uncoupled respiration does not exceed OXPHOS capacity in mouse samples. For mouse cerebellum, OXPHOS capacity decreased when comparing with maximal ETS capacity. LEAK respiration increased under physiologically controlled circumstances versus chemically induced (oligomycin). This could be due to the fact that endogenous available ADP is still phosphorylated to ATP, whereas with chemical induction, proton leak is maximal, resulting in an overestimation of LEAK respiration. In contrast to the cerebellum, the soleus was tested at hyperoxic conditions to avoid oxygen diffusion limitation and shows three times higher OXPHOS capacity. NADH-dependent respiration is different when analyzing specific types of tissue, with the soleus having more capacity to respire through the addition of succinate than the cerebellum. Both types of tissue show minimal ROX.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63000/63000fig5v2.jpg" /> 
Figure 5: Representative traces of oxygen consumption for non-fibrous (A) and fibrous (B) tissues for cHRR. (A) Wet weight tissue-normalized oxygen consumption trace of mouse cerebellum prepared as described (step 1.3). (B) Wet weight-tissue-normalized oxygen consumption trace of mouse soleus muscle as described (step 1.4). Blue line shows respective oxygen concentration and injection points. Abbreviations: see Figure 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63000/63000fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>BSA, fatty acid free</td>
			<td>1 g/L</td>
		</tr>
		<tr>
			<td>D-sucrose</td>
			<td>110 mM</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Lactobionic&#160;acid</td>
			<td>60 mM</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>3 mM</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>20 mM</td>
		</tr>
	</tbody>
</table>
Table 1: Mitochondrial respiration medium MiR05 composition adjusted to pH 7.127.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>CaK2EGTA anhydrous</td>
			<td>2.77 mM</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>K2EGTA, anhydrous</td>
			<td>7.23 mM</td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>MgCl2-6H2O</td>
			<td>6.56 mM</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>5.77 mM</td>
		</tr>
		<tr>
			<td>Na2Phosphocreatine</td>
			<td>15 mM</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>20 mM</td>
		</tr>
	</tbody>
</table>
Table 2: Relaxing and biopsy preservation solution (BIOPS) composition adjusted to pH 7.128.

Watch this Scientific Journal Video about High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers at JoVE.com

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and ...

high-resolution-respirometry-to-assess-bioenergetics-cells-tissues

Nanyang Technological University

Research

JoVE Journal

Biology

4.5K Views.  University of Helsinki. Mitochondriën leveren cellulaire energie door oxidatieve fosforylering die bijdraagt aan bijna alle processen in de cel. Bijgevolg worden mitochondriale disfuncties geassocieerd met een breed spectrum van ziekten. Het belangrijkste voordeel van deze techniek is de realtime meting van mitochondriale bio-energetica door zuurstofverbruik, en dus een nauwkeurige beoordeling van het totale cellulaire energiemetabolisme.Respirometrie met hoge resolutie maakt een directe beoordeling mogelijk...

Video: High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

Analyzing Mitochondrial Physiology and Bioenergetics in Fruit Flies

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Author Spotlight: Exploring Mitochondrial Function and Chemical Toxicity Using Drosophila melanogaster 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

Biochemistry

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Bioengineering

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Author Spotlight: Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...

Immunology and Infection

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Determining Respiration in Human Sperm Cells by High-Resolution Respirometry

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

Author Spotlight: Advancing Male Infertility Research by Unraveling Sperm Metabolism and Mitochondrial Function 

The analysis of sperm mitochondrial function by high-resolution respirometry permits the measurement of the oxygen consumption of freely moving spermatozoa in a closed-chamber system. The technique can be applied to measure respiration in human spermatozoa, which provides information on sperm mitochondrial characteristics and integrity.

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm ...

Medicine

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Cellular Respiration

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Quantifying Respiration using Microrespirometers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To assemble the microrespirometers needed for the lab, first plug in the hot glue gun to preheat it.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Push the plunger of a tuberculin syringe all the way in and then cut the end off of the syringe.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, insert a capillary tube into the empty needle end of the syringe.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Push the capillary tube in until it touches the tip of the plunger.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Use the hot glue gun to create an airtight seal between the capillary tube and the tuberculin syringe. Make sure that the capillary tube is standing straight up while the glue dries.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Once the glue has dried, pull back on the plunger and make sure the capillary tube is not plugged.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Next, prepare the manometer fluid by adding red food coloring to soapy water. NOTE: The detergent coats the inside of the capillary tube, preventing the manometer fluid from sticking during the experiment.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Insert the capillary tube into the manometer fluid and then pull the plunger on the syringe until the entire capillary tube has been filled.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, eject the manometer fluid back out of the microrespirometer.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">With a small piece of absorbent cotton, plug the barrel of the microrespirometer, inserting it into the syringe up to the 0 mL mark. Pack the cotton using a spare capillary tube or a glass disposable pipette.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Add a single drop of 15% KOH to the cotton using a spare capillary tube or a glass disposable pipette.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Add some nonabsorbent cotton to the KOH saturated cotton to act as a barrier to the seeds. Pack the cotton using a disposable pipette. The cotton should reach halfway between the 0 mL and 1 mL mark.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Reinsert the plunger carefully until it reaches the cotton and then remove any excess KOH by pressing it out.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Set four microrespirometers at each lab station.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Then, place a lab marker and two small dishes containing the seeds and glass beads for each group alongside the microrespirometers.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Finally, prepare two water baths by filling them with roughly 6 inches of water. The water level in the bath should comfortably fit two respirometers with the entirety of their two capillary tubes exposed.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Replace the lids and insert a thermometer in each bath to monitor the temperature. One bath should be set at about 25 &#176;C, and the other at approximately 40 &#176;C.
</div></li></ul></li></ol>

Cellular Respiration: Quantification using Microrespirometers - Prep

Quantifying Respiration using Microrespirometers
ExpandTo assemble the microrespirometers needed for the lab, first plug in the hot glue gun to preheat ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

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