Name: A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation (Video) | JoVE Journal
Uploaded: 2018-07-13T13:00:00.000+00:00
Duration: 7 min 14 s
Description: Learn a flow cytometry-based JC-1 assay to measure mitochondrial membrane potential in human cardiac myocytes after hypoxia/reoxygenation. This efficient method evaluates protective agents against cellular injury with low material requirements.

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5 minutes

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 (for more details, see11). Store at 4 &#176;C and warm to 37 &#176;C before using.</li>
	<li>Prepare 5 mL of JC-1 working solution by adding 25 &#181;L of JC-1 stock solution (200x), 4 mL of ultrapure water, and 1 mL of stock staining buffer (5x) in a 15 mL centrifuge tube in the dark. Pipette the mixture up and down several times and warm to 37 &#176;C before using.</li>
	<li>Prepare 30 mL of working staining buffer by adding 24 mL of ultrapure water to 6 mL of stock staining buffer (5x) in a 50 mL centrifuge tube. Use this solution ice-cold.</li>
</ol>
2. Establishment of Hypoxia/Reoxygenation Model
<ol>
	<li>Plate 1.0 x 105 cells per well with HCM complete medium in a 6 well plate. Grow to reach a confluency of about 70% in a cell incubator containing 5% CO2 and 95% air at 37 &#176;C.</li>
	<li>Wash HCMs with phosphate buffer saline (PBS). Expose them to different treatments (400 &#181;g/mL TXL or not) in 2 mL of serum/glucose-free DMEM for 30 min prior to hypoxia in a cell incubator containing 5% CO2 and 95% air at 37 &#176;C.</li>
	<li>Incubate HCMs in an airtight and hypoxic jar (2.5 L) fitted with a catalyst to scavenge free oxygen and induce hypoxia for 18 h (Figure 1A), and an anaerobic indicator to measure the oxygen tension in the medium11,12,13, in a cell incubator containing 5% CO2 and 95% air at 37 &#176;C. 
	Note: It takes about 1.5 h for the catalyst to scavenge free oxygen in the jar. Therefore, the HCMs are kept in the jar for 19.5 h before the jar is opened. The color of anaerobic indicator changes from light blue in normoxic environment to pale white in hypoxic condition (Figure 1B).</li>
	<li>Reoxygenate HCMs by moving the 6 well plate into an incubator set at 37 &#176;C containing 5% CO2 and 95% air for 2 h.</li>
</ol>
3. Preparation of HCMs for Flow Cytometry
<ol>
	<li>Warm the 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution and PBS to 37 &#176;C.</li>
	<li>Aspirate medium from each well, rinse the cells with 1 mL of PBS and add 0.5 mL of 0.25% trypsin along the wall of the well.</li>
	<li>Incubate at 37 &#176;C until all HCMs are almost detached (about 1 min).</li>
	<li>Add 1 mL of DMEM complete medium (DMEM medium with 10% fetal bovine serum) to the wells to neutralize the trypsin. Transfer the cell suspension to a 15 mL centrifuge tube and pellet HCMs by centrifuging at 500 x g for 3 min at room temperature.</li>
	<li>Carefully aspirate the supernatant without disturbing the pellet.</li>
	<li>Add 0.5 mL of HCM complete medium and 0.5 mL of JC-1 working solution to each tube. Resuspend the HCMs by pipetting up and down.</li>
	<li>Cover the whole tubes with aluminum foil to prevent bleaching of the fluorescent indicator and incubate HCMs in normal conditions (at 37 &#176;C containing 5% CO2 and 95% air) for 20 min.</li>
	<li>Pellet HCMs by centrifuging at 500 x g for 3 min at 4 &#176;C.</li>
	<li>Rinse HCMs twice with 2 mL of ice-cold JC-1 staining buffer.</li>
	<li>Resuspend HCMs in a final volume of 300 &#181;L of ice-cold staining buffer in the sample tube (12 mm x 75 mm) and use the cells for analysis within 30 min. 
	Note: Use carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a potent inhibitor of respiratory electron transport chain, to treat HCMs for 1 h at a concentration of 20 &#181;M, for the positive control.</li>
</ol>
4. Flow Cytometer Setup
<ol>
	<li>Start the flow cytometer and ensure that the software is connected to it.</li>
	<li>Perform fluidics startup, start the stream and set up the breakoff.</li>
	<li>Open two dot plot windows in the flow cytometry software.
	<ol>
		<li>Select forward scattered light (FSC) on the X axis and side scattered light (SSC) on the Y axis in the first dot plot to represent the characteristics of the cells in terms of their size and their granularity, respectively.</li>
		<li>Select the PE (575 &#177; 26 nm) detection channel for the Y axis and FITC (530 &#177; 30 nm) for the X axis using a logarithmic scale in the second dot plot, which is used to measure the fluorescence intensity of JC-1 dye in the cells.</li>
	</ol>
	</li>
</ol>
5. Flow Cytometry Measurement and Data Analysis
<ol>
	<li>Click Unload to let out the tube support arm and position the Blank (HCMs that have not been dyed with JC-1) sample tube on it. Click Load to return the tube support arm to its working position.</li>
	<li>Click Record to collect particles from suspension in the Blank sample tube and then gate the cell population (P1) for further analysis in the first dot plot (Figure 3).</li>
	<li>Collect HCMs in other sample tubes and optimize the measurement by adjusting the voltages of fluorescence channels, to make sure that the cell dot is located in the central area of the second dot plot.</li>
	<li>Display the statistics of each sample tube and calculate the MMPs of each sample by dividing the ratio of red fluorescence intensity by that of green fluorescence intensity.</li>
</ol>

<ol><li>Anderson, J. L., Morrow, D. A. <a target="_blank" href="http://www.nejm.org/doi/full/10.1056/NEJMra1606915">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/pubmed?term=((ESC+Guidelines+for+the+management+of+acute+myocardial+infarction+in+patients+presenting+with+ST-segment+elevation%3A+The+Task+Force+for+the+management+of+acute+myocardial+infarction+in+patients+presenting+with+ST-segment+elevation+of+the+European+Society+of+Cardiology+%28ESC%29%5BTitle%5D)+AND+%22European+Heart+Journal%22%5BJournal%5D)">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/pubmed?term=((Myocardial+reperfusion+injury%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">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/pubmed?term=((The+mitochondrial+permeability+transition+pore+and+its+role+in+myocardial+ischemia+reperfusion+injury%5BTitle%5D)+AND+%22Journal+of+Molecular+and+Cellular+Cardiology%22%5BJournal%5D)">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/pubmed?term=((Molecular+basis+of+cardioprotection%3A+signal+transduction+in+ischemic+pre-%2C+post-%2C+and+remote+conditioning%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">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/pubmed?term=((Functional+heterogeneity+of+an+isolated+mitochondrial+population+revealed+by+cytofluorometric+analysis+at+the+single+organelle+level%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">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/pubmed?term=((Mitochondrial+membrane+potential+and+glutamate+excitotoxicity+in+cultured+cerebellar+granule+cells%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Mitochondrial+membrane+potential+probes+and+the+proton+gradient%3A+a+practical+usage+guide%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">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/pubmed?term=((Flow+cytometric+analysis+of+mitochondrial+membrane+potential+using+JC-1%5BTitle%5D)+AND+%22Current+Protocols+in+Cytometry%22%5BJournal%5D)">Flow cytometric analysis of mitochondrial membrane potential using JC-1.</a> Current Protocols in Cytometry. , Chapter 9 Unit 9 14(2001).</li>
<li>Salvioli, S., Ardizzoni, A., Franceschi, C., Cossarizza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((JC-1%2C+but+not+DiOC6%283%29+or+rhodamine+123%2C+is+a+reliable+fluorescent+probe+to+assess+delta+psi+changes+in+intact+cells%3A+implications+for+studies+on+mitochondrial+functionality+during+apoptosis%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">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/pubmed?term=((Inhibition+of+miR-128-3p+by+Tongxinluo+Protects+Human+Cardiomyocytes+from+Ischemia%2Freperfusion+Injury+via+Upregulation+of+p70s6k1%2Fp-p70s6k1%5BTitle%5D)+AND+%22Frontiers+in+Pharmacology%22%5BJournal%5D)">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/pubmed?term=((Induction+of+autophagy+by+Tongxinluo+through+the+MEK%2FERK+pathway+protects+human+cardiac+microvascular+endothelial+cells+from+hypoxia%2Freoxygenation+injury%5BTitle%5D)+AND+%22Journal+of+Cardiovascular+Pharmacology%22%5BJournal%5D)">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/pubmed?term=((Lysophosphatidic+acid+protects+mesenchymal+stem+cells+against+hypoxia+and+serum+deprivation-induced+apoptosis%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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://cshprotocols.cshlp.org/content/2011/9/pdb.prot065490.short">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/pubmed?term=((Anesthetic-induced+preconditioning+delays+opening+of+mitochondrial+permeability+transition+pore+via+protein+Kinase+C-epsilon-mediated+pathway%5BTitle%5D)+AND+%22Anesthesiology%22%5BJournal%5D)">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/pubmed?term=((Suppression+of+Excessive+Histone+Deacetylases+Activity+in+Diabetic+Hearts+Attenuates+Myocardial+Ischemia%2FReperfusion+Injury+via+Mitochondria+Apoptosis+Pathway%5BTitle%5D)+AND+%22Journal+of+Diabetes+Research%22%5BJournal%5D)">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/pubmed?term=((Curcumin-induced+melanoma+cell+death+is+associated+with+mitochondrial+permeability+transition+pore+%28mPTP%29+opening%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">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/pubmed?term=((P53+dependent+mitochondrial+permeability+transition+pore+opening+is+required+for+dexamethasone-induced+death+of+osteoblasts%5BTitle%5D)+AND+%22Journal+of+Cell+Physiology%22%5BJournal%5D)">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/pubmed?term=((Nox2+as+a+potential+target+of+mitochondrial+superoxide+and+its+role+in+endothelial+oxidative+stress%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">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/pubmed?term=((Effect+of+mitochondrial+KATP+channel+on+voltage-gated+K%2B+channel+in+24+hour-hypoxic+human+pulmonary+artery+smooth+muscle+cells%5BTitle%5D)+AND+%22Chinese+Medical+Journal+%28Engl%29%22%5BJournal%5D)">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/pubmed?term=((The+Neuroprotective+Effects+of+Hypothermia+on+Bilirubin-Induced+Neurotoxicity+in+vitro%5BTitle%5D)+AND+%22Neonatology%22%5BJournal%5D)">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/pubmed?term=((Autophagy+activation+by+Jiang+Zhi+Granule+protects+against+metabolic+stress-induced+hepatocyte+injury%5BTitle%5D)+AND+%22World+Journal+of+Gastroenterology%22%5BJournal%5D)">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/pubmed?term=((Renoprotective+Effects+of+Aldose+Reductase+Inhibitor+Epalrestat+against+High+Glucose-Induced+Cellular+Injury%5BTitle%5D)+AND+%22Biomed+Research+International%22%5BJournal%5D)">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/pubmed?term=((Mouse+Liver+Mitochondria+Isolation%2C+Size+Fractionation%2C+and+Real-time+MOMP+Measurement%5BTitle%5D)+AND+%22Bio-Protocols%22%5BJournal%5D)">Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement.</a> Bio-Protocols. 6 (15), (2016).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>Mitochondrial membrane potential assay kit with JC-1</td><td>Beyodtime, China</td><td>C2006</td><td>In the kit there are JC-1 stock solution (200&amp;times;), stock staining buffer (5&amp;times;) and CCCP(10mM)</td></tr><tr><td>Tongxinluo ultrafine powder</td><td>Shijiazhuang Yiling Pharmaceutical Co., China</td><td>071201</td><td></td></tr><tr><td>Annexin V-FITC/PI Kit</td><td>Becton-Dickinson, USA</td><td>556547</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies, Grand Island Biological Company, USA</td><td>11966-025</td><td></td></tr><tr><td>Human cardiac myocyte</td><td>Promocell, Germany</td><td>C-12810</td><td></td></tr><tr><td>Myocyte Growth Medium&lt;br/&gt; (SupplementMix)</td><td>Promocell, Germany</td><td>C-39275</td><td></td></tr><tr><td>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><td>GENbox</td><td>BioM&amp;eacute;rieux, Marcy l&amp;rsquo;Etoile, France</td><td>96127</td><td>2.5L</td></tr><tr><td>Catalyst (AnaeroPack)</td><td>MITSUBISHI GAS CHEMICAL&amp;nbsp;COMPANY, INC.&amp;nbsp;, Japan</td><td>&amp;nbsp;C-1</td><td></td></tr><tr><td>Anaerobic indicator</td><td>BioM&amp;eacute;rieux, Marcy l&amp;rsquo;Etoile, France</td><td>96118</td><td></td></tr><tr><td>Flow cytometer</td><td>Becton-Dickinson, USA</td><td>FACSAria 2</td><td></td></tr><tr><td>BD FACSDiva Software</td><td>Becton-Dickinson, USA</td><td></td><td>Version8.0.1</td></tr><tr><td>Sample tube</td><td>Corning science, USA</td><td>352054</td><td>12*75mm</td></tr><tr><td>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

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±), indicating that we had established a cell-based model of I/R (45.00 ± 2.13% vs. 11.50 ± 0.18% in the no...

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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Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

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

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

Learn a flow cytometry-based JC-1 assay to measure mitochondrial membrane potential in human cardiac myocytes after hypoxia/reoxygenation. This efficient method evaluates protective agents against cellular injury with low material requirements.

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

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