Name: high-resolution respirometry to assess mitochondrial function in permeabilized and intact cells
Uploaded: 2017-02-08T13:00:00
Description: Watch this Scientific Journal Video about High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells at JoVE.com

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

1. Cell Culture
<ol>
	<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...

<ol>
	<li>Brand, M. D., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochem J. 435 (2), 297-312 (2011).</li><li>Lanza, I. R., Nair, K. 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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 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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Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

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 ...

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or enzymes are involved in the pathogenesis of several diseases. Mitochondrial substrate flux can be studied using intact cells, permeabilized cells, or isolated mitochondria. Investigating intact cells encounters several problems due to simultaneous oxidation of different substrates. Besides, several cell types contain internal stores of different substrates that complicate results interpretation. Methods such as mitochondrial isolation or using permeabilizing agents are not easily reproducible. Isolating pure mitochondria with intact membranes in sufficient amounts from small samples is problematic. Using non-selective permeabilizers causes various degrees of unavoidable mitochondrial membrane damage. Recombinant perfringolysin O (rPFO) was offered as a more appropriate permeabilizer, thanks to its ability to selectively permeabilize plasma membrane without affecting mitochondrial integrity. When used in combination with microplate respirometry, it allows testing the flux of several mitochondrial substrates with enough replicates within one experiment while using a minimal number of cells. In this work, the protocol describes a method to compare mitochondrial substrate flux of two different cellular phenotypes or genotypes and can be customized to test various mitochondrial substrates or inhibitors.

In this work, we describe a modified protocol to test mitochondrial respiratory substrate flux using recombinant perfringolysin O in combination with microplate-based respirometry. With this protocol, we show how metformin affects mitochondrial respiration of two different tumor cell lines.

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or ...

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.

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 ...

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 ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...