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>
	<li>Anderson, J. L., Morrow, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Myocardial+Infarction.">Acute Myocardial Infarction.</a> New England Journal of Medicine. 376 (21), 2053-2064 (2017).</li><li>Ibanez, B., 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=ESC+Guidelines+for+the+management+of+acute+myocardial+infarction+in+patients+presenting+with+ST-segment+elevation:+The+Task+Force+for+the+management+of+acute+myocardial+infarction+in+patients+presenting+with+ST-segment+elevation+of+the+European+Society+of+Cardiology+(ESC).">ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC).</a> European Heart Journal. 39 (2), 119-177 (2017).</li><li>Yellon, D. M., Hausenloy, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+reperfusion+injury.">Myocardial reperfusion injury.</a> New England Journal of Medicine. 357 (11), 1121-1135 (2007).</li><li>Ong, S. B., Samangouei, P., Kalkhoran, S. B., Hausenloy, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+permeability+transition+pore+and+its+role+in+myocardial+ischemia+reperfusion+injury.">The mitochondrial permeability transition pore and its role in myocardial ischemia reperfusion injury.</a> Journal of Molecular and Cellular Cardiology. 78, 23-34 (2015).</li><li>Heusch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+cardioprotection:+signal+transduction+in+ischemic+pre-,+post-,+and+remote+conditioning.">Molecular basis of cardioprotection: signal transduction in ischemic pre-, post-, and remote conditioning.</a> Circulation Research. 116 (4), 674-699 (2015).</li><li>Cossarizza, A., Ceccarelli, D., Masini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+an+isolated+mitochondrial+population+revealed+by+cytofluorometric+analysis+at+the+single+organelle+level.">Functional heterogeneity of an isolated mitochondrial population revealed by cytofluorometric analysis at the single organelle level.</a> Experimental Cell Research. 222 (1), 84-94 (1996).</li><li>Ward, M. W., Rego, A. C., Frenguelli, B. G., 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=Mitochondrial+membrane+potential+and+glutamate+excitotoxicity+in+cultured+cerebellar+granule+cells.">Mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells.</a> Journal of Neuroscience. 20 (19), 7208-7219 (2000).</li><li>Perry, S. W., Norman, J. P., Barbieri, J., Brown, E. B., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+potential+probes+and+the+proton+gradient:+a+practical+usage+guide.">Mitochondrial membrane potential probes and the proton gradient: a practical usage guide.</a> Biotechniques. 50 (2), 98-115 (2011).</li><li>Cossarizza, A., Salvioli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+mitochondrial+membrane+potential+using+JC-1.">Flow cytometric analysis of mitochondrial membrane potential using JC-1.</a> Current Protocols in Cytometry. , 14 (2001).</li><li>Salvioli, S., Ardizzoni, A., Franceschi, C., Cossarizza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JC-1,+but+not+DiOC6(3)+or+rhodamine+123,+is+a+reliable+fluorescent+probe+to+assess+delta+psi+changes+in+intact+cells:+implications+for+studies+on+mitochondrial+functionality+during+apoptosis.">JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis.</a> FEBS Letters. 411 (1), 77-82 (1997).</li><li>Chen, G. H., 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=Inhibition+of+miR-128-3p+by+Tongxinluo+Protects+Human+Cardiomyocytes+from+Ischemia/reperfusion+Injury+via+Upregulation+of+p70s6k1/p-p70s6k1.">Inhibition of miR-128-3p by Tongxinluo Protects Human Cardiomyocytes from Ischemia/reperfusion Injury via Upregulation of p70s6k1/p-p70s6k1.</a> Frontiers in Pharmacology. 8, 775 (2017).</li><li>Cui, H., 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=Induction+of+autophagy+by+Tongxinluo+through+the+MEK/ERK+pathway+protects+human+cardiac+microvascular+endothelial+cells+from+hypoxia/reoxygenation+injury.">Induction of autophagy by Tongxinluo through the MEK/ERK pathway protects human cardiac microvascular endothelial cells from hypoxia/reoxygenation injury.</a> Journal of Cardiovascular Pharmacology. 64 (2), 180-190 (2014).</li><li>Chen, J., 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=Lysophosphatidic+acid+protects+mesenchymal+stem+cells+against+hypoxia+and+serum+deprivation-induced+apoptosis.">Lysophosphatidic acid protects mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis.</a> Stem Cells. 26 (1), 135-145 (2008).</li><li>Chazotte, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+mitochondria+with+JC-1.">Labeling mitochondria with JC-1.</a> Cold Spring Harbor Protocols. (9), (2011).</li><li>Pravdic, D., 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=Anesthetic-induced+preconditioning+delays+opening+of+mitochondrial+permeability+transition+pore+via+protein+Kinase+C-epsilon-mediated+pathway.">Anesthetic-induced preconditioning delays opening of mitochondrial permeability transition pore via protein Kinase C-epsilon-mediated pathway.</a> Anesthesiology. 111 (2), 267-274 (2009).</li><li>Wu, Y., 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=Suppression+of+Excessive+Histone+Deacetylases+Activity+in+Diabetic+Hearts+Attenuates+Myocardial+Ischemia/Reperfusion+Injury+via+Mitochondria+Apoptosis+Pathway.">Suppression of Excessive Histone Deacetylases Activity in Diabetic Hearts Attenuates Myocardial Ischemia/Reperfusion Injury via Mitochondria Apoptosis Pathway.</a> Journal of Diabetes Research. 2017, 8208065 (2017).</li><li>Qiu, Y., 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=Curcumin-induced+melanoma+cell+death+is+associated+with+mitochondrial+permeability+transition+pore+(mPTP)+opening.">Curcumin-induced melanoma cell death is associated with mitochondrial permeability transition pore (mPTP) opening.</a> Biochemical and Biophysical Research Communications. 448 (1), 15-21 (2014).</li><li>Zhen, Y. F., 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=P53+dependent+mitochondrial+permeability+transition+pore+opening+is+required+for+dexamethasone-induced+death+of+osteoblasts.">P53 dependent mitochondrial permeability transition pore opening is required for dexamethasone-induced death of osteoblasts.</a> Journal of Cell Physiology. 229 (10), 1475-1483 (2014).</li><li>Nazarewicz, R. R., Dikalova, A. E., Bikineyeva, A., Dikalov, S. 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. J. <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+mitochondrial+KATP+channel+on+voltage-gated+K++channel+in+24+hour-hypoxic+human+pulmonary+artery+smooth+muscle+cells.">Effect of mitochondrial KATP channel on voltage-gated K+ channel in 24 hour-hypoxic human pulmonary artery smooth muscle cells.</a> Chinese Medical Journal (Engl). 118 (1), 12-19 (2005).</li><li>Kuter, N., Aysit-Altuncu, N., Ozturk, G., Ozek, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Neuroprotective+Effects+of+Hypothermia+on+Bilirubin-Induced+Neurotoxicity+in+vitro.">The Neuroprotective Effects of Hypothermia on Bilirubin-Induced Neurotoxicity in vitro.</a> Neonatology. 113 (4), 360-365 (2018).</li><li>Zheng, Y. Y., Wang, M., Shu, X. B., Zheng, P. Y., Ji, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+activation+by+Jiang+Zhi+Granule+protects+against+metabolic+stress-induced+hepatocyte+injury.">Autophagy activation by Jiang Zhi Granule protects against metabolic stress-induced hepatocyte injury.</a> World Journal of Gastroenterology. 24 (9), 992-1003 (2018).</li><li>El Gamal, H., Eid, A. H., Munusamy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renoprotective+Effects+of+Aldose+Reductase+Inhibitor+Epalrestat+against+High+Glucose-Induced+Cellular+Injury.">Renoprotective Effects of Aldose Reductase Inhibitor Epalrestat against High Glucose-Induced Cellular Injury.</a> Biomed Research International. 2017, 5903105 (2017).</li><li>Renault, T. T., Luna-Vargas, M. P., Chipuk, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Liver+Mitochondria+Isolation,+Size+Fractionation,+and+Real-time+MOMP+Measurement.">Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement.</a> Bio-Protocols. 6 (15), (2016).</li></ol>

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.

a-flow-cytometry-based-assay-for-measuring-mitochondrial-membrane

Research

JoVE Journal

Medicine

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6 Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8 
This video demonstrates a protocol for MMT, which has been taught to &ge;43 research staff who have performed &gt;800 assessments on &gt;280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11 Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2.

Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale.

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory rate, salivation, perspiration and renal function. The ANS has two main branches: the sympathetic nervous system, preparing the human body for action in times of danger and stress, and the parasympathetic nervous system, which regulates the resting state of the body.
ANS activity can be measured invasively, for instance by radiotracer techniques or microelectrode recording from superficial nerves, or it can be measured non-invasively by using changes in an organ's response as a proxy for changes in ANS activity, for instance of the sweat glands or the heart. Invasive measurements have the highest validity but are very poorly feasible in large scale samples where non-invasive measures are the preferred approach. Autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram (ECG) in combination with the impedance cardiogram (ICG), which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta. From the respiration and ECG signals, respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control. From the ECG and the left ventricular ejection signals, the preejection period can be extracted as a measure of cardiac sympathetic control. ECG and ICG recording is mostly done in laboratory settings. However, having the subjects report to a laboratory greatly reduces ecological validity, is not always doable in large scale epidemiological studies, and can be intimidating for young children. An ambulatory device for ECG and ICG simultaneously resolves these three problems.
Here, we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children, using a validated ambulatory device 1-5, the VU University Ambulatory Monitoring System (VU-AMS, Amsterdam, the Netherlands,<a href="http://www.vu-ams.nl" target="_blank"> www.vu-ams.nl</a>).

Measuring Cardiac Autonomic Nervous System ANS Activity in Children

Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

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 responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

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 body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.