Name: a flow cytometry-based assay for measuring mitochondrial membrane potential in cardiac myocytes after hypoxia/reoxygenation
Uploaded: 2018-07-13T13:00:00
Description: Watch this Scientific Journal Video about A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation at JoVE.com

This study was supported by grants from The National Key Research and Development Program of China (No. 2017YFC1700503), the National Basic Research Program (973 Program) of China (No.2012CB518602), the National Natural Science Foundation of China (No. 81370223 and No. 81573957), and the Postgraduates&#39; Innovative Research Foundation of Peking Union Medical College (2016-1002-01-02).

1. Preparation of Reagents and Solutions
<ol>
	<li>Prepare human cardiac myocytes (HCMs) complete medium by adding 25 mL of Myocyte Growth Medium supplement mix to 500 mL of Myocyte Growth Medium as per the manufacturer&#8217;s instructions. Store at 4 &#176;C and warm to 37 &#176;C before using.</li>
	<li>Prepare Tongxinluo (TXL) Solution by dissolving TXL ultrafine powder in serum/glucose-free Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) and adjust the concentration of TXL to 400 &#181;g/mL by adding DMEM (f...

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nox2+as+a+potential+target+of+mitochondrial+superoxide+and+its+role+in+endothelial+oxidative+stress.">Nox2 as a potential target of mitochondrial superoxide and its role in endothelial oxidative stress.</a> American Journal of Physiology-Heart and Circulatory Physiology. 305 (8), H1131-H1140 (2013).</li><li>Wang, T., Zhang, Z. X., Xu, Y. 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Here, we present a protocol that uses JC-1 dye to assess the MMP of cells after being exposed to H/R. Detected by JC-1 assay, the MMP of cells is independent of factors such as mitochondrial size, shape, and density that may influence single-component fluorescence signals14. Consequently, the results of JC-1 assay are relatively reliable. Besides, it is convenient and time-saving to perform the JC-1 assay. This assay has a low requirement for materials and reagents, and generally, it takes less th...

Coronary heart disease is the leading cause of death worldwide. The treatment of choice for reducing ischemic injury and limiting infarct size in patients with ST-segment elevated myocardial infarction is timely and effective myocardial reperfusion via primary percutaneous coronary intervention (PCI)1,2. However, reperfusion causes additional damage, which can account for up to 30 percent of the final infarct size3. It is universally acknowledged that the mitochondrial permeability transition pore (mPTP) is not only central in mitochondrial damage and cell death during ischemia/reperfusion (I/R), but is also a converging target of cardioprotective signaling4,5. As the mPTP opening would bring about depolarization of the inner mitochondrial membrane potential (MMP)4, we detected mPTP opening using the 5,5&#8242;,6,6&#8242;-Tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-imidacarbocyanineiodide (JC-1) assay.
The JC-1 assay is a cytofluorimetric method that is both qualitative and quantitative, and it has been further validated by analyzing the MMP at the level of a single mitochondria6. JC-1 exists as an aggregated form, yielding a red to orange colored emission (590 &#177; 17.5 nm) in the matrix of mitochondria with normal MMP; with the loss of MMP, JC-1 is converted to the monomeric form that yields green fluorescence with an emission of 530 &#177; 15 nm. Therefore, a decrease in the red/green fluorescence intensity ratio could indicate the reduction of MMP in conditions such as ischemia/reperfusion (I/R).
In addition to JC-1, MMP has also been studied with membrane-permeable lipophilic cations such as Rhodamine 123 and 3,3&#8242;-dihexyloxadicarbocyanine iodide [DiOC6(3)]. However, compared to these two probes, JC-1 is more reliable for analyzing MMP. Rhodamine 123 has relatively poor sensitivity (especially in quenching mode7,8) and poor specificity. The shift in rhodamine 123 sometimes is so small that it is hard for researchers or equipment to observe/detect. Besides, in a single cell, there are different mitochondrion binding sites for rhodamine 123 and so that it may have different fluorescence emissions9. DiOC6(3) is not recommended for detecting MMP either as it reacts sensitively to the depolarization of plasma membrane10.
Therefore, here we use the JC-1 assay to assess the MMP of HCMs after being exposed to hypoxia/reoxygenation with or without a protective agent.

<table border="0" cellpadding="0" cellspacing="0" width="970">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Mitochondrial membrane potential assay kit with JC-1</td>
			<td style="width:307px;">Beyodtime, China</td>
			<td style="width:80px;">C2006</td>
			<td style="width:291px;">In the kit there are JC-1 stock solution (200&#215;), stock staining buffer (5&#215;) and CCCP(10mM)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tongxinluo ultrafine powder</td>
			<td>Shijiazhuang Yiling Pharmaceutical Co., China</td>
			<td>071201</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Annexin V-FITC/PI Kit</td>
			<td>Becton-Dickinson, USA</td>
			<td>556547</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM</td>
			<td>Life Technologies, Grand Island Biological Company, USA</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human cardiac myocyte</td>
			<td>Promocell, Germany</td>
			<td>C-12810</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Myocyte Growth Medium 
			(SupplementMix)</td>
			<td>Promocell, Germany</td>
			<td>C-39275</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Myocyte Growth Medium (Ready-to-use)</td>
			<td>Promocell, Germany</td>
			<td>C-22070</td>
			<td>used with Myocyte Growth Medium SupplementMix</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GENbox</td>
			<td>BioM&#233;rieux, Marcy l&#8217;Etoile, France</td>
			<td>96127</td>
			<td>2.5L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Catalyst (AnaeroPack)</td>
			<td>MITSUBISHI GAS CHEMICAL&#160;COMPANY, INC.&#160;, Japan</td>
			<td>&#160;C-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anaerobic indicator</td>
			<td>BioM&#233;rieux, Marcy l&#8217;Etoile, France</td>
			<td>96118</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow cytometer</td>
			<td>Becton-Dickinson, USA</td>
			<td>FACSAria 2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD FACSDiva Software</td>
			<td>Becton-Dickinson, USA</td>
			<td></td>
			<td>Version8.0.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sample tube</td>
			<td>Corning science, USA</td>
			<td>352054</td>
			<td>12*75mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>Hyclone, USA</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow cytometry-based assay for measuring mitochondrial membrane potential in cardiac myocytes after hypoxia/reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Before performing the JC-1 assay to evaluate the changes of MMP, it is highly recommended that experiments be carried out to confirm the conditions successfully set by the researchers. As shown by the flow cytometry results (Figure 2), compared with the normal group, hypoxia/reoxygenation (H/R) significantly induced the apoptosis of HCMs (Annexin V + /PI&#177;), indicating that we had established a cell-based model of I/R (45.00 &#177; 2.13% vs. 11.50 &#177; ...

Watch this Scientific Journal Video about A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation at JoVE.com

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

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