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.).

All animal experimentation is performed in accordance with the National Animal Experiment Review Board and Regional State Administrative Agency for Southern Finland. Male C57BL/6JOlaHsd mice (4-6 months-old) were used in this study. Consent for the use of human cell lines was obtained from the institutional ethics committee of the University of Helsinki.
1. High-resolution respirometry: Chamber-based respirometer (cHRR)
NOTE: The experiments in this s...

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, discu...

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/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/files/ftp_upload/63000/63000fig02large.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>2 mL Potter-Elvehjem Glass/PTFE Tissue Grinder/Homogenizer</td>
			<td>Omni International</td>
			<td>07-358029</td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2, 5% CO2 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>CaCO3</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&#8217;s modified Eagle&#8217;s medium (High glucose)</td>
			<td>Fisher Scientific</td>
			<td>41965-039</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium&#160;(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&#160;(100x)&#160;(200 nM&#160;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>MgCl2</td>
			<td>Sigma</td>
			<td>M 9272</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>Sigma</td>
			<td>A 2383</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Na2Phosphocreatine</td>
			<td>Sigma</td>
			<td>P 7936</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Na-pyruvate&#160;(100 mM)&#160;(100x)</td>
			<td>Fisher Scientific</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA&#160;(Non-essential amino acids) 100x</td>
			<td>Fisher Scientific</td>
			<td>11140035</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal FBS&#160;(10x)</td>
			<td>Fisher Scientific</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>O2k-Core: Oxygraph-2k</td>
			<td>Oroboros Instruments</td>
			<td>10000-02</td>
			<td>High-resolution respirometry instrument</td>
		</tr>
		<tr>
			<td>O2k-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> 
			Seahorse XF DMEM assay medium pack, pH 7.4</td>
			<td> 
			Agilent, Santa Clara, CA</td>
			<td>103680-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Extracellular Flux Analyzer</td>
			<td> 
			Agilent, Santa Clara, CA</td>
			<td></td>
			<td>High-throughput respirometry instrument</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td> 
			Agilent, Santa Clara, CA</td>
			<td></td>
			<td> 
			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 mHR...

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 ...

Mitochondria provide cellular energy through oxidative phosphorylation contributing to almost all processes within the cell. Consequently, mitochondrial dysfunctions are associated with a wide spectrum of diseases. The main advantage of this technique is the realtime measurement of mitochondrial bioenergetics by oxygen consumption, and thus, an accurate assessment of total cellular energy metabolism.High-resolution respirometry allows direct assessment of what in the oxidative phosphorylation system is failing, potentially providing diagnostic clues in primary mitochondrial diseases and secondary mitochondrial dysfunctions associated with many disorders. Demonstrating the procedure will be Ryan Awadhpersad, PhD student in my lab. To begin, perform the oxygen calibration by running the respirometers in 2.1 milliliters of mitochondrial respiration medium at 37 degrees Celsius for more than 45 minutes and proceed if baseline variation is within four picomoles per second.Culture HEK293 cells in high-glucose DMEM supplemented with 10%heat-activated FBS, GlutaMAX, non-essential amino acids, sodium pyruvate, and uridine in an incubator at 37 degrees Celsius at 5%carbon dioxide. On the day of the experiment, collect and count the cells and resuspend 2.5 million cells in 2.5 milliliters respiration medium. To assess routine respiration, add 2.3 milliliters of warm respiration medium cell suspension to the chamber.Run the chambers at 37 degrees Celsius and a stirring speed of 700 RPM. Wait for at least three minutes to allow media to de-gas, then close the chambers by twisting the stopper in a rotating motion. Next, aspirate the excess liquid on top of the stopper.After 10 minutes, obtain a stable oxygen flux signal to record routine or state I respiration. To perform OXPHOS analysis in intact cells, add two microliters of 0.01-millimolar oligomycin for a final concentration of 10-nanomolar. Titrate FCCP from two-millimolar stock by adding 0.6 microliters at 0.2-microliter steps until no increase in respiration is observed and respiration is maximally uncoupled.Next, add one microliter of one-millimolar rotenone for a final concentration of 0.5-micromolar. Then, add two microliters of one milligram per milliliter antimycin stock for a final concentration of one microgram per milliliter. Reoxygenate the chamber to the same oxygen level, approximately 150-micromolar, by lifting the plunger.Afterward, add five microliters of 0.8-molar ascorbate for a final concentration of two-millimolar. Then, immediately add five microliters of 0.2-molar TMPD for a final concentration of 0.5-millimolar and evaluate complex IV activity. As the peak oxygen flux is reached with TMPD, add five microliters or four-molar azide for a final concentration of 10-millimolar.Then, continue the run for at least five minutes to assay auto-oxidation of TMPD for complex IV base level calculation. After the run, collect one milliliter of sample suspension from each chamber and centrifuge at 1, 000 G for permeabilized cells or at 20, 000 G for tissue lysate. Then, discard the supernatant and freeze the pellet at minus 80 degrees Celsius for further downstream analysis.First, seed the cells according to the growth rates of individual cell lines. Dilute oligomycin, FCCP, rotenone, and antimycin in three microliters of assay medium to a final concentration of 1.5-micromolar, 1.125-micromolar, and one-micromolar, respectively. Subsequently, fill them into separate ports.Observe the injection ports to ensure an even loading of the samples. Turn on the microplate-based system and computer and equilibrate them at 37 degrees Celsius for at least three hours. On the day of the microplate-based assay, verify the confluency of the cell culture plate, the morphology of cells, and ensure that the background wells are empty.Then, remove everything except 20 microliters of the culture medium from each well. Then, wash each well with 90 microliters of assay medium and add 100 microliters of assay medium to make a final volume of up to 120 microliters. Next, incubate the plate at 37 degrees Celsius in an incubator without carbon dioxide for 60 minutes.After retrieving the plate from the incubator, remove the lid and place the microplate in the slot. Click on Continue to start the run. After the run is finished, take the plate out and remove the remaining assay media without disturbing the cells.Then, freeze the entire plate at minus 80 degrees Celsius. After performing the digitonin permeabilization and respiration experiments, raw oxygen consumption traces of wild-type HEK293 cells and HEK293 cells with a CRISPR-mediated gene knockout resulting in multiple OXPHOS deficiencies were recorded. Overlaid cell input-normalized oxygen consumption traces were obtained.CRISPR knockout 1 shows impaired respiration and CRISPR knockout 2 shows no respiration compared to wild-type when normalized to cell number. Protein amounts were quantified and respiration values were normalized to protein amount in order to determine absolute values of respiratory states and respective flux control ratios. Extracellular acidification rates were found to increase for OXPHOS deficiency in CRISPR knockout 2, suggesting compensation of mitochondrial oxidative phosphorylation deficiency in HEK293 cells through increased glycolysis.In addition, the respiration values were determined in the presence of oligomycin, FCCP, and rotenone, and the values obtained were normalized to the protein amount. Protein-normalized oxygen consumption traces of wild-type HEK293 cells and HEK293 cells with CRISPR-mediated mitochondrial translation deficiency causing multiple OXPHOS deficiency was studied. Wet weight tissue-normalized oxygen consumption traces of mouse cerebellum and mouse soleus muscle were obtained.The soleus muscles show three times higher OXPHOS and respiration capacity than the cerebellum. With chamber-based respirometry, it is important to work fast, as mitochondrial function will decline the moment you collect your samples, and with plate-based respirometry, it is crucial to acquire an optimal seeding density to minimize variability. For further analysis, protein quantification or immuno-blotting is possible.This could determine whether an alteration in mitochondrial respiratory function was due to abundance of OXPHOS complexes or mitochondrial amount. Already a century ago, cancer cells were found to perform anaerobic glycolysis in addition to mitochondrial OXPHOS. This underlines the need to assay mitochondrial bioenergetics.Here we demonstrated two respirometers considered today's gold standard.

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

Research

JoVE Journal

Biology

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 ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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 ...

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...

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, ...