The authors thank the staff members of the Department of Physiology in the Faculty of Medicine in Hradec Kr&#225;lov&#233; and the Department of Pathophysiology in the Third Faculty of Medicine for the help with chemicals and samples preparation. This work was supported by Charles University grant programs PROGRES Q40/02, Czech Ministry of Health grant NU21-01-00259, Czech science foundation grant 18-10144 and INOMED project CZ.02.1.01/0.0/0.0/18_069/0010046 funded by the Ministry of Education, Youth and Sports of the Czech Republic and by the European Union.

1. One day before the assay
<ol>
	<li>Preparation of reagents and substrates.
	<ol>
		<li>Mitochondrial assay solution (MAS): Prepare stock solutions of all reagents as described in Table 1. Warm the stocks of mannitol and sucrose to 37 &#176;C to dissolve completely. Mix the reagents to prepare 2x MAS, then warm the mixture to 37 &#176;C. Adjust the pH with 5N KOH to 7.4 (~7 mL), then add water to bring the volume up to 1 L. Filter-sterilize&#160;and store the aliquots at -20 &#176;C until the measure...

<ol>
	<li>Gerencser, A. A., 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=Quantitative+microplate-based+respirometry+with+correction+for+oxygen+diffusion.">Quantitative microplate-based respirometry with correction for oxygen diffusion.</a> Analytical Chemistry. 81 (16), 6868-6878 (2009).</li><li>Murphy, E., 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=Mitochondrial+function,+biology,+and+role+in+disease:+A+scientific+statement+from+the+American+Heart+Association.">Mitochondrial function, biology, and role in disease: A scientific statement from the American Heart Association.</a> Circulation Research. 118 (12), 1960-1991 (2016).</li><li>Owen, O. E., Kalhan, S. C., Hanson, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+key+role+of+anaplerosis+and+cataplerosis+for+citric+acid+cycle+function.">The key role of anaplerosis and cataplerosis for citric acid cycle function.</a> Journal of Biological Chemistry. 277 (34), 30409-30412 (2002).</li><li>Nicholls, D. G., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioenergetics 3. , (2002).</li><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> Biochemical Journal. 435 (2), 297-312 (2011).</li><li>Sta&#328;kov&#225;, P., 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=Adaptation+of+mitochondrial+substrate+flux+in+a+mouse+model+of+nonalcoholic+fatty+liver+disease.">Adaptation of mitochondrial substrate flux in a mouse model of nonalcoholic fatty liver disease.</a> International Journal of Molecular Sciences. 21 (3), 1101 (2020).</li><li>Salabei, J. K., Gibb, A. A., Hill, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+measurement+of+respiratory+activity+in+permeabilized+cells+using+extracellular+flux+analysis.">Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.</a> Nature Protocols. 9 (2), 421-438 (2014).</li><li>Divakaruni, A. S., 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=Thiazolidinediones+are+acute,+specific+inhibitors+of+the+mitochondrial+pyruvate+carrier.">Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (14), 5422-5427 (2011).</li><li>Divakaruni, A. S., Rogers, G. W., Murphy, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+function+in+permeabilized+cells+using+the+seahorse+XF+analyzer+or+a+Clark-type+oxygen+electrode.">Measuring mitochondrial function in permeabilized cells using the seahorse XF analyzer or a Clark-type oxygen electrode.</a> Current Protocols in Toxicology. 60, 1-16 (2014).</li><li>Elkalaf, M., T&#367;ma, P., Weiszenstein, M., Pol&#225;k, J., Trnka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+probe+Methyltriphenylphosphonium+(TPMP)+inhibits+the+Krebs+cycle+enzyme+2-Oxoglutarate+dehydrogenase.">Mitochondrial probe Methyltriphenylphosphonium (TPMP) inhibits the Krebs cycle enzyme 2-Oxoglutarate dehydrogenase.</a> PLoS One. 11 (8), 0161413 (2016).</li><li>Rogers, G. W., 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=High+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria.">High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.</a> PLoS One. 6 (7), 21746 (2011).</li></ol>

This protocol is a modification of previously published studies7,8,9,10 and the product user guide. In contrast to the manufacturer&#39;s protocol, 2x MAS is used instead of 3x MAS, since 2&#215; MAS is easier to dissolve and does not form precipitations after freezing. Frozen 2x MAS aliquots can be stored up to six months and show consistent results. Another difference is including ADP in the ...

Microplate-based respirometry has revolutionized mitochondrial research by enabling the study of cellular respiration of a small sample size1. Cellular respiration is generally considered as an indicator of mitochondrial function or &#39;dysfunction&#39;, despite the fact that the mitochondrial range of functions extends beyond energy production2. In aerobic conditions, mitochondria extract the energy stored in different substrates by breaking down and converting these substrates into metabolic intermediates that can fuel the citric acid cycle3 (Figure 1). The continuous flux of substrates is essential for the flow of the citric acid cycle to generate high energy &#39;electron donors&#39;, which deliver electrons to the electron transport chain that generates a proton gradient across the inner mitochondrial membrane, enabling ATP-synthase to phosphorylate ADP to ATP4. Therefore, an experimental design to assay mitochondrial respiration must include the sample nature (intact cells, permeabilized cells, or isolated mitochondria) and mitochondrial substrates.
Cells keep a store of indigenous substrates5, and mitochondria oxidize several types of substrates simultaneously6, which complicates the interpretation of results obtained from experiments performed on intact cells. A common approach to investigate mitochondrial ability to oxidize a selected substrate is to isolate mitochondria or permeabilize the investigated cells5. Although isolated mitochondria are ideal for quantitative studies, the isolation process is laborious. It faces technical difficulties such as the need for large sample size, purity of the yield, and reproducibility of the technique5. Permeabilized cells offer a solution for the disadvantages of mitochondrial isolation; however, routine permeabilizing agents of detergent nature are not specific and may damage mitochondrial membranes5.
Recombinant perfringolysin O (rPFO) was offered as a selective plasma membrane permeabilizing agent7, and it was used successfully in combination with an extracellular flux analyzer in several studies7,8,9,10. We have modified a protocol using rPFO to screen mitochondrial substrate flux using XFe96 extracellular flux analyzer. In this protocol, four different substrate oxidizing pathways in two cellular phenotypes are compared while having sufficient replicates and the proper control for each tested material.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adinosine 5&#8242; -diphosphate monopotassium salt dihydrate</td>
			<td>Merck</td>
			<td>A5285</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Merck</td>
			<td>A8674</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Merck</td>
			<td>A3803</td>
			<td>store at 2 - 8 &#176;C</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td>Merck</td>
			<td>C2920</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Merck</td>
			<td>D8418</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Merck</td>
			<td>63559</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid</td>
			<td>Merck</td>
			<td>03777</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Merck</td>
			<td>H7523</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>L(-)Malic acid disodium salt</td>
			<td>Merck</td>
			<td>M9138</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>L-Glutamic acid sodium salt hydrate</td>
			<td>Merck</td>
			<td>G5889</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Magnissium chloride hexahydrate</td>
			<td>Merck</td>
			<td>M2670</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Merck</td>
			<td>O4876</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Palmitoyl-DL-carnitine chloride</td>
			<td>Merck</td>
			<td>P4509</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Merck</td>
			<td>484016</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Merck</td>
			<td>P5655</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Merck</td>
			<td>R8875</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Seahorse Wave Desktop Software</td>
			<td>Agilent technologies</td>
			<td></td>
			<td>Download from www.agilent.com</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent technologies</td>
			<td>102416-100</td>
			<td>XFe96 sensor cartridges and XF96 cell culture microplates</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Merck</td>
			<td>P2256</td>
			<td>store at 2 - 8 &#176;C</td>
		</tr>
		<tr>
			<td>Sodium succinate dibasic hexahydrate</td>
			<td>Merck</td>
			<td>S2378</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Merck</td>
			<td>S7903</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Merck</td>
			<td>W3500</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>XF calibrant</td>
			<td>Agilent technologies</td>
			<td>100840-000</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>XF Plasma membrane permeabilizer</td>
			<td>Agilent technologies</td>
			<td>102504-100</td>
			<td>Recombinant perfringolysin O (rPFO) - Aliquot and store at -20 &#176;C</td>
		</tr>
	</tbody>
</table>

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.

Start by normalizing the results to the second measurement of baseline respiration to show values as oxygen consumption rate percentage (OCR%). The results of the assay are shown in Figures 5, Figure 6, Figure 7, and Figure 8. It is important to assign the proper background wells for each group and inactivate the background wells of other groups. Fi...

Watch this Scientific Journal Video about Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells at JoVE.com

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

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 ...

Because it tests mitochondria substrate flux that can be affected with different pathologies or treatments. For example, today we will use it to reveal cancer cell response to Metformin treatment. It requires a minimal number of cells while having sufficient replicates and appropriate control for each distinct material.The analyzer is for research use only. This technique can identify errors in mitochondrial metabolism and can be used to evaluate warriors drugs affecting mitochondria. Demonstrating the procedure will be Karolina Vaneckova an undergraduate student and research technician from my laboratory.To begin seed A549 cells at a density of 20, 000 cells per well in columns, two to 11 of a seahorse XF 96 cell culture micro-plate. Leaving columns one and 12 empty as background wells. Fill the blank Wells with an equal volume of the cell culture medium, then incubate the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for cell attachment.After three to four hours, add 100 microliters of cell culture medium in the Wells. Treat the experimental group columns seven to 11 with one millimolar Metformin and the control group with an equal volume of sterile distilled water, then return the plate to the incubator. For hydrating the sensors, pipette 200 microliters of sterile water per well into the utility plate.Then carefully return the sensor cartridge while immersing the sensor in water. Incubate the cartridge in a carbon dioxide free incubator at 37 degrees Celsius till the next day. Switch on the analyzer and the control unit.Start the instrument control and data acquisition software, and design the assay protocol as described in the text manuscript. Under group definitions, create four injection strategies where port A differs according to the injected substrate and name the strategies after the substrates or abbreviations. Assign Oligomycin to port B, FCCP to port C and Rotenone Antimycin A mixture to port D.Create and name eight groups, under the plate map, assign groups to the corresponding Wells.Then save the protocol as a ready to use template. Leave the analyzer switch on to allow the temperature to stabilize overnight. The next day, discard the water from the utility plate and add 200 microliters of the pre-warmed calibrant per well in the utility plate.Return the cartridge to the carbon dioxide free incubator until the time of the assay. Maintain a source of humidity inside the incubator and turn off or reduce the fan speed to a minimum, to avoid rapid evaporation of the calibrant. Prepare five milliliters of the working solution of the substrates and inhibitors with pre-warmed two times mitochondrial assay solution, 5%BSA and sterile water as described in the text manuscript.Next, load the injector ports with substrate and inhibitors as described in the tax manuscript. For calibration under the run assay tab, click on the start run to start the assay. Insert the loaded censor cartridge and wait for the calibration to complete.Prepare a 20 milliliters of assay medium, by mixing two times mitochondrial assay solution, sterile water and 5%BSA in a 50 milliliter tube. Add two microliters of 10 micromolar recombinant perfringolysin O, to attain a concentration of one nanomolar. And re-suspend the mixture with gentle pipetting, avoiding shaking and vortexing.Incubate the tube at 37 degrees Celsius until use. Use a multi-channel pipette to wash the cells and the empty blank Wells two times, using pre-warmed calcium and magnesium free PBS solution. Discard the PBS and add 180 microliters of the pre-warmed assay medium for cell permeabilization.Immediately after permeabilization replace the utility plate of the calibrated sensor cartridge with the cell plate containing permeabilized cells and start the measurement. The treatment group had a higher rate of succinate induced respiration. The response of A549 cells to Metformin treatment was higher than Hep G2.For the pyruvate malate induced respiration, A549 cells showed increased induction compared to Hep G2 cells between five to 15 minutes.Similar results were obtained for glutamate malate induced respiration and palmitoylcarnitine malate induced respiration. To attain the right concentration of substrate and inhibitors. The right volume must be loaded into the right port.When a change in a metabolic pathway of a certain substrate is identified, it will be necessary to assess the function of the enzymes and transporters involved in that pathway.

measuring-mitochondrial-substrate-flux-recombinant-perfringolysin-o

Research

JoVE Journal

Biology

2.3K vistas.  Charles University. Porque prueba el flujo de sustrato mitocondrial que puede verse afectado con diferentes patologías o tratamientos. Por ejemplo, hoy lo usaremos para revelar la respuesta de las células cancerosas al tratamiento con metformina. Requiere un número mínimo de células mientras tiene suficientes réplicas y el control apropiado para cada material distinto.El analizador es solo para uso de investigación. Esta técnica puede identificar errores en el metabolismo mitocondrial y se puede u...