This study was supported by the Swiss National Science Foundation (Grant n&#186; 32003B_127619).

1. Cell Culture
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	<li>Culture human hepatoma HepG2 cells6 in 25 cm2 cell culture flasks in Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 &#176;C in an incubator (5% CO2, 95% air) (seeding density: 1 x 106 cells per 25 cm2 cell culture flask, incubation time in the 37 &#176;C incubator: 48 hr, cell density at confluency: 4-5 x 106 cells per 2...

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+remifentanil+on+mitochondrial+oxygen+consumption+of+cultured+human+hepatocytes.">Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes.</a> PLoS One. 7 (9), e45195 (2012).</li><li>Horan, M. P., Pichaud, N., Ballard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+quantifying+mitochondrial+dysfunction+in+complex+diseases+of+aging.">Review: quantifying mitochondrial dysfunction in complex diseases of aging.</a> J Gerontol A Biol Sci Med Sci. 67 (10), 1022-1035 (2012).</li><li>Niklas, J., Melnyk, A., Yuan, Y., Heinzle, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+permeabilization+for+the+high-throughput+measurement+of+compartmented+enzyme+activities+in+mammalian+cells.">Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells.</a> Anal Biochem. 416 (2), 218-227 (2011).</li><li>Jeger, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose+response+of+endotoxin+on+hepatocyte+and+muscle+mitochondrial+respiration+in+vitro.">Dose response of endotoxin on hepatocyte and muscle mitochondrial respiration in vitro.</a> Biomed Res Int. 2015, 353074 (2015).</li><li>Nicholls, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+c+binding+to+enzymes+and+membranes.">Cytochrome c binding to enzymes and membranes.</a> Biochim Biophys Acta. 346 (3-4), 261-310 (1974).</li><li>Cortese, J. D., Voglino, A. L., Hackenbrock, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+conformations+of+physiological+membrane-bound+cytochrome+c.">Multiple conformations of physiological membrane-bound cytochrome c.</a> Biochemistry. 37 (18), 6402-6409 (1998).</li><li>Gorbenko, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+cytochrome+c+complexes+with+phospholipids+as+revealed+by+resonance+energy+transfer.">Structure of cytochrome c complexes with phospholipids as revealed by resonance energy transfer.</a> Biochim Biophys Acta. 1420 (1-2), 1-13 (1999).</li><li>Gnaiger, E., M&#233;ndez, G., Hand, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+phosphorylation+efficiency+and+depression+of+uncoupled+respiration+in+mitochondria+under+hypoxia.">High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia.</a> Proc Natl Acad Sci USA. 97, 11080-11085 (2000).</li><li>Pesta, D., Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+respirometry:+OXPHOS+protocols+for+human+cells+and+permeabilized+fibers+from+small+biopsies+of+human+muscle.">High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle.</a> Methods Mol Biol. 810, 25-58 (2012).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Pathways+and+Respiratory+Control.+An+Introduction+to+OXPHOS+Analysis.">Mitochondrial Pathways and Respiratory Control. 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The authors have nothing to disclose.

The objective of the present protocol was to use high-resolution respirometry to measure mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity and mitochondrial outer membrane integrity.
There are some critical steps within the present protocol. First, cellular oxygen consumption rates are usually normalized to the number of cells (pmol/[sec x number of cells]). Therefore, before monitoring cellular oxygen consumption,...

Mitochondria fulfill important roles in cellular energy metabolism, especially by using oxygen to produce adenosine triphosphate (ATP). They are implicated in cell death and in several human diseases. Mitochondrial oxidative phosphorylation (OXPHOS) combines electron transport along the electron transport chain with oxygen consumption and ATP synthesis. The mitochondrial tricarboxylic acid (TCA) cycle is involved in the conversion of proteins, carbohydrates and fats into energy rich compounds as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). Electrons of the NADH and FADH2 are then transferred to the respiratory electron transport chain protein complexes (I to IV) located in the inner mitochondrial membrane. In addition, two other redox pathways can transfer electrons to electron transport chain: i) mitochondrial electron-transferring flavoprotein (ETF) which is located on the matrix face of the inner mitochondrial membrane, and supplies electrons from fatty acid &#946;-oxidation; and ii) mitochondrial glycerophosphate dehydrogenase which oxidizes glycerophosphate to dihydroxyacetone phosphate and feeds electrons to the mitochondrial electron transport chain. Complex IV (the ultimate electron acceptor) transfers the electrons to one oxygen molecule, converting oxygen to two molecules of water. Moving of the electrons from respiratory electron transport chain complex I to IV is coupled with proton flow from the mitochondrial matrix to the intermembrane space which establishes an electrochemical gradient across the mitochondrial inner membrane. Afterwards, mitochondrial complex V (ATP synthase) shuttles the hydrogen ions back into the mitochondrial matrix and synthesizes ATP molecules. OXPHOS function can be assessed in vivo and in vitro using various techniques and various mitochondrial respiration states can be obtained. In isolated mitochondria the following respiratory states can be measured: i) basal mitochondrial respiration (state 1), ii) oxygen consumption after the addition of specific substrates of the mitochondrial respiratory chain complexes (state 2), iii) maximal mitochondrial oxygen consumption after the addition of saturating concentrations of adenosine diphosphate (ADP) (state 3) and, iv) resting respiration after ADP consumption (converted to ATP) (state 4). In intact cells the following respiratory states can be measured: i) basal cellular oxygen consumption in the presence of endogenous substrates and ADP, ii) basal cellular oxygen consumption in the presence of oligomycin (oligomycin-insensitive respiration) and oligomycin-sensitive respiration (ATP turnover), iii) FCCP uncoupled respiration, and iv) non-mitochondrial respiration after the addition of antimycin A and rotenone. In permeabilized cells, specific substrates of the electron transport chain complexes and ADP can be added and maximal complex-dependent respiratory rates such as complex I-, II- and IV-dependent respiratory rates can be measured.
Measurements of cellular respiration provide important insights into mitochondrial respiratory capacity specific to complexes I-IV, mitochondrial integrity and energy metabolism1,2,3. One of the devices which enable measurements of mitochondrial oxygen consumption with high accuracy, resolution and sensitivity is the high-resolution oxygraph4. The high-resolution oxygraph device contains two chambers with injection ports and each chamber is equipped with a polarographic oxygen sensor. Cellular or isolated mitochondrial suspensions are stirred continuously in the respirometer. To assess mitochondrial function, substrates and inhibitors for mitochondrial complex activity can be added following standard protocols. Substrates and inhibitors can be titrated by injection into the chambers of the oxygraph, and oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. High-resolution respirometry offers several advantages over traditional and conventional polarographic oxygen electrode devices including increased sensitivity and the ability to work with small numbers of biological samples such as intact or permeabilized cells. In addition, each device contains two chambers, and respiratory rates can be recorded simultaneously for comparisons of oxygen concentrations. The high-resolution oxygraph also has the advantage of reduced leakage of oxygen from the device chambers compared to traditional polarographic oxygen electrode devices. Another device recently developed to measure cellular oxygen consumption is the 96-well extracellular flux analyzer5. The extracellular flux analyzer is equipped with fluorescence instead of polarographic sensors. The advantages of the extracellular flux analyzer compared to the high-resolution oxygraph are i) it is a largely automated device, ii) it is possible to measure oxygen consumption in 24- and 96-well plates for high-throughput screening, therefore requiring lower amounts of biological samples, and iii) additional measurement of cellular glycolytic flux is possible. The disadvantages of the extracellular flux analyzer in comparison to the high-resolution oxygraph are i) the high costs of the device and of consumables such as fluorescent plates, which are non-reusable, and ii) only four compounds per assay/well are injectable, therefore the system is not feasible for substrate-uncoupler-inhibitor titration protocols.
In the present study, we use high-resolution respirometry to determine mitochondrial respiration. For cellular oxygen consumption experiments, the cells are permeabilized to allow the entry of exogenous ADP and oxidizable mitochondrial substrates for feeding electrons into complexes of the respiratory system. This approach allows the dissection of individual mitochondrial complexes respiratory capacities, which is a distinct advantage compared to intact cells (many substrates are cell-impermeant). However, cell membrane permeabilization will disrupt the barrier between the cytosol and extracellular space and medium (wash out of cytosolic solutes) and the composition of the intracellular space is equilibrated with the extracellular medium. One of the disadvantages of permeabilized cells over intact cells is that the mitochondrial outer membrane can be damaged if excessive amounts of detergent are employed during cell permeabilization. In intact cells, basal, coupled and uncoupled respiration of intact cells can be measured. This method evaluates oxygen consumption of intact cells without the addition of exogenous substrates and ADP, reproducing the respiratory function in the integrated cell and also providing information on maximal mitochondrial electron transport capacity6,7. One of the advantages of intact cells over permeabilized cells is that cellular environment is not disrupted and mitochondria are in contact with the whole components of the cells. In order to permeabilize the cellular plasma membrane, detergents such as digitonin have been used8. However, mitochondrial outer membrane integrity can be compromised if excessive amounts of digitonin are employed. To confirm that mitochondrial outer membrane integrity is not compromised in permeabilized cells, digitonin titration is performed to determine the optimal concentration for cellular permeabilization. For these experiments, cells are resuspended in respiration medium and digitonin concentration is titrated by respirometry in the presence of mitochondrial substrates and ADP, and respiration rates are measured. Respiration of intact, non-permeabilized cells is not stimulated in the presence of mitochondrial substrates and ADP. However, subsequent stepwise digitonin titration would yield gradual permeabilization of plasma membranes, and optimal digitonin concentration is obtained. This is shown by the increase of respiration up to full permeabilization. Mitochondrial quality and outer membrane integrity can be verified by adding exogenous cytochrome c2,9. Cytochrome c is a 12 kDa electron carrying protein of the mitochondrial electron transport chain 10,11,12. It is localized in the mitochondrial intermembrane space, and is involved in oxygen consumption, carrying electrons from complex III to complex IV. Once the mitochondrial outer membrane is damaged, cytochrome c is released, and mitochondrial oxygen consumption is reduced. Upon addition of exogenous cytochrome c, any augmentation in mitochondrial respiration is indicative of a disrupted mitochondrial outer membrane.
In permeabilized cells, substrates and inhibitors of mitochondrial complex activity are added following various protocols3,9. For example in order to investigate mitochondrial complex-driven respiratory rates, the following protocol can be used. After permeabilization of the cells, first complex I is stimulated by the substrates malate and glutamate, which generate NADH as a substrate to the respiratory chain and provoke the activation of complex I. Afterwards, ADP is added for conversion to ATP (state 3, active complex I-dependent respiration). After a stable signal is reached, rotenone (mitochondrial complex I inhibitor) is administered to inhibit complex I. Rotenone is followed by succinate to FADH2 and to activate complex II (state 3, active complex II-dependent respiration). In order to measure complex IV-dependent respiration, first complex III-dependent respiration is inhibited by adding antimycin A (mitochondrial complex III inhibitor). Afterwards, complex IV-dependent respiration is stimulated by administering ascorbate and tetramethylphenylendiamine (TMPD). TMPD can auto-oxidize in the respiration buffer, therefore the maximal complex IV-dependent respiration rate (State 3) is calculated by subtracting respiration rates before and after the addition of sodium azide, an inhibitor of mitochondrial complex IV. The respiration experiments can be carried out in two chambers of an oxygraph in parallel-one serving as a control (unstimulated cells), the other containing the stimulated cells. Obviously, the cells can be pre-treated in various ways, e.g., with drugs affecting mitochondrial functions, before their oxygen consumption is measured in the oxygraph chamber. This protocol allows functional examination of the individual mitochondrial respiratory chain complexes. In addition, one can measure maximal ADP-stimulated respiration (state 3) of permeabilized cells, using exogenous fatty acid in the form of palmitate. In this protocol, concentrated stocks of sodium palmitate are conjugated with ultra fatty acid free bovine serum albumin (BSA) (6:1 molar ratio palmitate:BSA). Afterwards, cells first are permeabilized with digitonin and mitochondrial respiration is assessed by the addition of carnitine and palmitate followed by addition of ADP (state 3, maximal respiration). Then, oligomycin is added to mimic state 4 (state 4o) and respiratory control ratio (RCR value) is calculated as state 3/state 4o. &#946;-oxidation promotes production of acetyl CoA (which enters in the TCA cycle) and generation of FADH2 and NADH, the electrons of which are passed to the electron transport chain by electron-transferring flavoprotein and &#946;-hydroxyacyl-CoA dehydrogenase. Mitochondria are at the center of fatty acid metabolism and described palmitate-BSA protocol can be used by researchers examining fatty acid oxidation. In intact cells, activators and inhibitors of mitochondrial complex activity are added following a different protocol6,9. For these experiments, first oxygen consumption of non-permeabilized cells in the absence of exogenous substrates is measured (phosphorylating respiration rate). Then, the non-phosphorylating respiration rate is measured after the addition of oligomycin, which is an inhibitor of mitochondrial ATP synthase. Afterwards, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) is administrated at various concentrations and the maximal mitochondrial uncoupled respiratory rate is measured. Protonophores such as FCCP can induce an augmentation in proton permeability of the inner membrane, allowing passive movement of protons to dissipate the chemiosmotic gradient. An increase in proton permeability uncouples oxidative respiration (no ATP production) and induces an increase in oxygen consumption. Afterwards, rotenone and antimycin A are added to inhibit mitochondrial respiration, and non-mitochondrial respiration is subtracted from all other respiratory rates.
The oxygen consumption rates can be expressed as IO2 [pmol x sec-1 x 10-6 cells] (oxygen flow per million cells) which is calculated by dividing volume-specific oxygen flux (in the closed oxygen chamber), JV,O2 [pmol x sec-1 x ml-1] by cell concentration in the cell chamber (number of cells per volume [106 cells&#8729;ml1])15. Cell-mass specific oxygen flux, JO2 [pmol x sec-1 x mg-1], is flow per cell, IO2 [pmol x sec-1 x 10-6 cells], divided by mass per cell [mg&#8729;106 cells]; or volume-specific flux, JV, O2 [pmol x sec-1 x ml-1], divided by mass per volume [mg&#8729;ml1]. JO2 is the oxygen flux per cell protein, dry weight or cell volume.
In the present study using high-resolution respirometry, we describe protocols to determine i) optimum digitonin concentration for complete cellular plasma membrane permeabilization (digitonin titration assay), ii) mitochondrial outer membrane integrity using exogenous cytochrome c, iii) mitochondrial respiratory chain complexes I, II and IV maximal respiratory rates in digitonin-permeabilized HepG2 cells in the presence of exogenous ADP and mitochondrial respiratory chain substrates, and iv) basal, coupled and maximal uncoupled respiration (maximal electron transport capacity) of intact cells without the addition of exogenous substrates and ADP, reproducing the respiratory function in the integrated cell.

<table border="0" cellpadding="0" cellspacing="0" width="415">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">ADP</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">A 4386</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:156px;">Antimycin A</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">A 8674</td>
			<td style="width:92px;">Chemical, dissolve in ethanol</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:156px;">Ascorbate</td>
			<td style="width:92px;">Merck</td>
			<td style="width:75px;">1.00127</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">BSA</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">A 6003</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">FCCP</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">C 2920</td>
			<td style="width:92px;">Chemical, dissolve in ethanol</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:156px;">Countess automated cell counter&#160;</td>
			<td style="width:92px;">Thermo Fisher Scientific</td>
			<td style="width:75px;">n/a</td>
			<td style="width:92px;">Automated cell counting instrument</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Cytochrome c</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">C 7752</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Digitonin</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">D 5628</td>
			<td style="width:92px;">Chemical, dissolve in DMSO</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">DMEM&#160;</td>
			<td style="width:92px;">Gibco</td>
			<td style="width:75px;">31966021</td>
			<td style="width:92px;">Medium</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">EGTA</td>
			<td style="width:92px;">fluka</td>
			<td style="width:75px;">3779</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">FBS</td>
			<td style="width:92px;">Gibco</td>
			<td style="width:75px;">26010-074</td>
			<td style="width:92px;">Medium component</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Glutamate</td>
			<td style="width:92px;">Sigma,</td>
			<td style="width:75px;">G 1626</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Hepes</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">H 7523</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">KCl</td>
			<td style="width:92px;">Merck</td>
			<td style="width:75px;">1.04936</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">KH2PO4</td>
			<td style="width:92px;">Merck</td>
			<td style="width:75px;">1.04873</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">K-lactobionate</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">L 2398</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">MgCl2</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">M 9272</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:156px;">O2k-Core: Oxygraph-2k&#160;</td>
			<td style="width:92px;">Oroboros Instruments</td>
			<td style="width:75px;">10000-02</td>
			<td style="width:92px;">High-resolution respirometry instrument</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Oligomycin</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">O 4876</td>
			<td style="width:92px;">Chemical, dissolve in ethanol</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Penicillin-streptomycin</td>
			<td style="width:92px;">Gibco</td>
			<td style="width:75px;">15140122</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Sodium azide</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">S2002</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Rotenone</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">R 8875</td>
			<td style="width:92px;">Chemical, dissolve in ethanol</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Succinate</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">S 2378</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">Taurine</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">T 8691</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:156px;">TMPD</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:75px;">T 3134</td>
			<td style="width:92px;">Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td style="width:92px;">Sigma</td>
			<td>T 4674</td>
			<td style="width:92px;">Chemical</td>
		</tr>
	</tbody>
</table>

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.

Determination of Optimum Digitonin Concentration for Cellular Permeabilization: Digitonin Titration Experiment
Digitonin titration is performed to determine the optimal concentration for permeabilization of HepG2 cells. For these experiments, digitonin is titrated in intact cells in the presence of rotenone, succinate (mitochondrial complex II substrate) and a saturating amount of ADP (to induce complex II-dependent state 3), and r...

Watch this Scientific Journal Video about High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells at JoVE.com

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

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 ...

The overall goal of this procedure is to use high-resolution respirometry to determine mitochondrial oxygen consumption. This method can help answer key questions in the syndromes and diseases associated with mitochondrial dysfunction such as;sepsis, diverse neurological diseases and age-related disorders. The main advantages of this technique is that it has higher sensitivity and the ability to perform substrate on coupled inhibitor titrations experiments with a small number of biological samples such as intact or permeabilized cells.Demonstrating the procedure will be Sandra Nansoz, a technician from my laboratory. Before beginning the procedure, air calibrate the polarographic oxygen sensors then resuspend the cells in respiration buffer to a one times ten to the sixth cells per milliliter concentration and replace the respiration medium in one chamber of the oxygraph with 2.1 milliliters of the cell suspension. Close the chamber with the stopper and set the magnetic stirring bar in the chamber to 700 rotations per minute.Then record the cellular respiration for 5 to 10 minutes until a stable oxygen flux signal is achieved. Next, use a syringe to inject two microliters of Rotenone through the titanium injection port into the oxygraph chamber and record the cellular respiration for another 5 to 10 minutes. When a stable oxygen flux signal is achieved, inject 20 microliters of one molar succinate followed by 10 microliters of 0.5 molar ADP.Then inject two volumes of two microliters of two millimolar digitonin and record the cellular respiration for two to five minutes after each injection followed by the step-wise injection of two to four microliters of two millimolar digitonin until the oxygen flux signal reaches the maximum level and further digitonin injections do not increase the respiration rate. For evaluation of the mitochondrial outer membrane integrity prepare an oxygraph chamber as just demonstrated and inject two microliters of eight millimolar digitonin to permeabilize the cells. After five minutes, inject 20 microliters of one molar succinate and record the cellular respiration for 5 to 10 minutes until a stable oxygen flux signal is achieved.Then inject 10 microliters of 0.5 molar ADP to stimulate complex two, and to induce an oxygen consumption increase. When a stable flux signal is achieved, inject five microliters of four millimolar cytochrome c followed by one microliter of four milligrams per milliliter of ligamycin. To measure the ADP-stimulated respiration of the liver hepatocellular carcinoma cells, prepare an oygraph chamber as just demonstrated, and inject two microliters of eight millimolar digitonin into the oxygen chamber to permeabilize the cells for five minutes, followed by the injection of 12.5 microliters of 0.8 molar mallade and 10 microliters of two molar glutamate.When a stable oxygen flux is achieved, inject 10 microliters of 0.5 molar ADP to increase the oxygen consumption, followed by two microliters of 0.2 millimolar rotenone and 20 microliters of one molar succinate. Next, inject two microliters of five millimolar antimycin and record the cellular respiration. When the signal decreases and stabilizes, inject 2.5 microliters of 0.8 millimolar escorbate followed by the immediate injection of 2.5 microliters of 0.2 millimolar TMPD, recording the cellular respiration until the oxygen flux signal increases and stabilizes.Then inject 10 microliters of one molar sodium azite into the oxygraph chamber, and record the cellular aspiration until the oxygen flux signal decreases and stabilizes. To measure the intact cell oxygen consumption, prepare an oxygraph chamber as just demonstrated and inject 1 microliter of four milligrams per milliliter aligimicyn followed by one and three microliter injections of 0.2 millimolar FCCP. Next, titrate the FCCP in 0.1 to 0.3 micromolar steps by injecting one to three microliters of 0.2 to one millimolar FCCP until the oxygen flux signal reaches its maximal level with no further increases, and then begins to decline.Then, inject two microliters of 0.2 millimolar rotenone and two microliters of five millimolar antimycin A and record the respiration until the oxygen flux signal decreases and stabilizes. In the absence of digitonin, cellular respiration is very low, and the respiration of intact, non-permeabilized cells is not stimulated in the presence of mitochondrial substrate and ADP. Upon stepwise addition of digitonin, however, the cellular plasma membrane becomes permeabilized and the mitochondrial respiration increases until full permeabilization, at which time the succinate and ADP enter the cells.Mitochondrial outer membrane integrity can be compromised, however, if excessive amounts of digitonin are employed, inducing a reduction in complex to dependent state three respiration. Cytochrome c does not enhance complex to dependent state three respiration in digitonin-treated cells, indicating that there is no loss of cytochrome c from the mitochondrial outer membrane, and that mitochondrial integrity is preserved, even though cytochrome c can enhance the respiration of cells treated with very high doses of digitonin. The addition of exogenous substrates of mitochondrial complex activity post-digitonin permeablization induces an increase in mitochondrial respiratory chain complexes one, two, and four maximal respiratory rates.The generation of reduced respiratory levels may be due to contamination of the oxygraph chambers with mitochondrial inhibitors from the previous experiment. In the presence of alygimycin and the sequential addition of FCCP, the maximal uncoupled respiratory rate of the cells is observed. Once mastered, any of these techniques can be done in less than one hour if it is performed properly.While attempting this procedure, it is very important to remember wash oxygraph chambers and stoppers extensively after each experiment. Following this procedure, other methods like measurements of cellular ATP content, each presentate in cymatic activity, mitochondrial substrate levels can be performed in order to answer additional questions like whether changes in respiratory rates are due to lack of mitochondrial substrates or reduced ATP cyntates activity. After its development, this technique paved the way for researchers in the field of bionegetics to explore mitochondrial function in fields such as acquired and genetic mitochondrial diseases, oxydative stress, skin perfusion injury and aging in intact or permeabilized cells or fibers and isolated mitochondria.After watching this video, you should have a good understanding of how to assist mitochondria function in permeabilized and intact cells using high-resolution respirometry. Don't forget that working with antimycin a, rotenone, sodium iozide, FCCP and aricomycin can be extremely dangerous and precautions such as wearing gloves and laboratory coats should always be taken while performing this procedure.

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41.0K Views.  University of Bern. The overall goal of this procedure is to use high-resolution respirometry to determine mitochondrial oxygen consumption. This method can help answer key questions in the syndromes and diseases associated with mitochondrial dysfunction such as;sepsis, diverse neurological diseases and age-related disorders. The main advantages of this technique is that it has higher sensitivity and the ability to perform substrate on coupled inhibitor titrations experiments with a small number of biological sa...