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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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 ...

This method can help to measure the effect of hypoxia/reoxygenation on cells in the mitochondrial membrane potential, and it can be used to evaluate if an agent is protective against a injury. The main advantage of this assay is that MMP detected by it is in the event of factors such as mitochondrial size. Additionally, it has a low requirement for materials and the radiance.Begin with 70%confluent human cardiac myocytes in a six well plate. After washing the cells with phosphate buffer saline, add drug or vehicle in two mililiters of serum and glucose free DMEM. Incubate in a cell culture incubator containing 5%CO2 and 95%air at 37 degrees Celsius for 30 minutes prior to hypoxia.After 30 minutes place the dish of human cardiac myocytes into a 2.5 liter airtight anhypoxic jar. Fitted with a catalyst to scavenge free oxygen, and an anerobic indicator to measure the oxygen tension in the medium. Place the jar in the tissue culture incubator.The color of the anerobic indicator changes from light blue in normoxic environment to pale white in hypoxic conditions. Reoxygenate the human cardiac myocytes by removing the six well plate from the jar and placing it on the incubator shelf for two hours. Following the reoxygenation, rinse the cells with one milliliter of pre-warmed PBS, and add 0.5 mililiters of pre-warmed 25%trypsin along the wall of the well.Incubate at 37 degrees Celsius for about one minute until all human cardiac myocytes are almost detached. Then add one milliliter of DMEM complete medium to the wells to neutralize the trypsin. Transfer the cell suspension to a 15 milliliter centrifuge tube, and pellet HCMs by centrifuging at 500 times G for three minutes, at room temperature.Following centrifugalization, carefully aspirate the supernatant without disturbing the pellets. Then add 5 milliliters of human cardiac myocyte complete medium, and 5 milliliters of JC-1 working solution to each tube. Resuspend the HCMs by pipetting up and down.Cover the tubes with aluminum foil to prevent bleaching of the fluorescent indicator, and incubate the cells in normal conditions for 20 minutes. After pelleting the human cardiac myocytes by centrifuging at 500 times G for three minutes in a centrifuge pre-cooled to four degrees Celsius, rinse cells twice with two milliliters of ice cold JC-1 staining buffer. After the second rinse, resuspend the human cardiac myocytes in 300 microliters of ice cold staining buffer.And filter the samples into the respective sample tubes. Analyze the cells by flow cytometry within 30 minutes. Start the flow cytometer and ensure that the software is connected.Perform fluidic start up. Start the stream, and set up the break off. Open two dot plot windows in the flow cytometry software.Select forward scattered light on the X-axis, and side scattered light on the Y-axis in the first dot plot. To represent the characteristics of the cells in terms of their size an their granularity, respectively. Select the PE detection channel for the Y-axis.And FITC for the X-axis, using a logarithmic scale in the second dot plot, which is used to measure the florescence intensity of JC-1 dye in the cells. Click unload to let out the tube support arm, and position the blank sample tube containing human cardiac myocytes that have not been dyed with JC-1. Click load to return the support arm to its working position.Click record to collect particles from suspension in the blank sample tube. And adjust voltages of flouresence channels in the second dot plot. Gate the cell population for further analysis in the first dot plot.Next, analyze the human cardiac myocytes. Display the statistics of each sample tube and calculate the MMPs of each sample by dividing the ratio of red flouresence intensity by that of green flouresence intensity. Hypoxia and reoxygenation significantly induce apoptosis of human cardiac myocytes, compared to normal cells as measured by the Annexin PI ratio.a Chinese traditional medicine with cardio-protective effects, prevented the apoptosis of HCMs after hypoxia/reoxygenation. As shown in here, the JC-1 red/green ratio after treatment with the mitochondrial CCP was almost zero. Consistent with the results from the apoptosis assay.The H/R treated group displayed a significantly lower red/green ratio, compared with the normal group. And reversed such a ratio change. While attempting this procedure, it's important to remember to start the cells loaded with JC-1 in ice cold standing buffer, as temperature has a considerable effect on the stabilities of mitochondrial membrane potential.Following this procedure, other methods like genetic manipulation can be performed to answer additional questions like if the decrease of MMP results from the opening of MPTP.

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Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...