Name: visualizing mitophagy with fluorescent dyes for mitochondria and lysosome
Uploaded: 2022-11-30T14:30:00
Description: Watch this Scientific Journal Video about Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome at JoVE.com

This work was partially funded by the National Key Research and Development Program of China (2017YFA0105601, 2018YFA0107102), the National Natural Science Foundation of China (81970333,31901044,), and the Program for Professor of Special Appointment at Shanghai Institutions of Higher Learning (GZ2020008).

1. Cell culture and passaging
NOTE: The protocol is described using routinely cultured mouse embryonic fibroblasts (MEFs) as an example.
<ol>
	<li>Culture MEF cells in 10 cm cell culture dishes with 10 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM). Incubate at 37 &#176;C and 5% CO2 and monitor the cells under a microscope at 100x magnification.</li>
	<li>Perform routine cell passaging.
	<ol>
		<li>When the cells reach 80%-90% confluency (every 3 days), wash th...

<ol>
	<li>Tao, M., 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=Animal+mitochondria:+evolution,+function,+and+disease.">Animal mitochondria: evolution, function, and disease.</a> Current Molecular Medicine. 14 (1), 115-124 (2014).</li><li>Henze, K., Martin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+biology:+essence+of+mitochondria.">Evolutionary biology: essence of mitochondria.</a> Nature. 426 (6963), 127-128 (2003).</li><li>Kiriyama, Y., Nochi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-+and+intercellular+quality+control+mechanisms+of+mitochondria.">Intra- and intercellular quality control mechanisms of mitochondria.</a> Cells. 7 (1), (2017).</li><li>Rossmann, M. P., Dubois, S. M., Agarwal, S., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+function+in+development+and+disease.">Mitochondrial function in development and disease.</a> Disease Models &amp; Mechanisms. 14 (6), 048912 (2021).</li><li>Suliman, H. B., Piantadosi, C. 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+quality+control+as+a+therapeutic+target.">Mitochondrial quality control as a therapeutic target.</a> Pharmacological Reviews. 68 (1), 20-48 (2016).</li><li>Wong, H. S., Dighe, P. A., Mezera, V., Monternier, P. A., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+superoxide+and+hydrogen+peroxide+from+specific+mitochondrial+sites+under+different+bioenergetic+conditions.">Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions.</a> Journal of Biological Chemistry. 292 (41), 16804-16809 (2017).</li><li>Zhao, R. Z., Jiang, S., Zhang, L., Yu, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+electron+transport+chain,+ROS+generation+and+uncoupling+(Review).">Mitochondrial electron transport chain, ROS generation and uncoupling (Review).</a> International Journal of Molecular Medicine. 44 (1), 3-15 (2019).</li><li>Murphy, M. P., Hartley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+as+a+therapeutic+target+for+common+pathologies.">Mitochondria as a therapeutic target for common pathologies.</a> Nature Reviews: Drug Discovery. 17 (12), 865-886 (2018).</li><li>Fan, 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=Mitochondrial+quality+control+in+cardiomyocytes:+a+critical+role+in+the+progression+of+cardiovascular+diseases.">Mitochondrial quality control in cardiomyocytes: a critical role in the progression of cardiovascular diseases.</a> Frontiers in Physiology. 11, 252 (2020).</li><li>Ma, K., 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=Mitophagy,+mitochondrial+homeostasis,+and+cell+fate.">Mitophagy, mitochondrial homeostasis, and cell fate.</a> Frontiers in Cell and Developmental Biology. 8, 467 (2020).</li><li>Kraus, F., Roy, K., Pucadyil, T. J., Ryan, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+and+regulation+of+the+divisome+for+mitochondrial+fission.">Function and regulation of the divisome for mitochondrial fission.</a> Nature. 590 (7844), 57-66 (2021).</li><li>Ren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics:+fission+and+fusion+in+fate+determination+of+mesenchymal+stem+cells.">Mitochondrial dynamics: fission and fusion in fate determination of mesenchymal stem cells.</a> Frontiers in Cell and Developmental Biology. 8, 580070 (2020).</li><li>Onishi, M., Yamano, K., Sato, M., Matsuda, N., Okamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+physiological+functions+of+mitophagy.">Molecular mechanisms and physiological functions of mitophagy.</a> The EMBO Journal. 40 (3), 104705 (2021).</li><li>Palikaras, K., Lionaki, E., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mitophagy+in+cellular+homeostasis,+physiology+and+pathology.">Mechanisms of mitophagy in cellular homeostasis, physiology and pathology.</a> Nature Cell Biology. 20 (9), 1013-1022 (2018).</li><li>Khaminets, A., Behl, C., Dikic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-dependent+and+independent+signals+in+selective+autophagy.">Ubiquitin-dependent and independent signals in selective autophagy.</a> Trends in Cell Biology. 26 (1), 6-16 (2016).</li><li>Fritsch, L. E., Moore, M. E., Sarraf, S. A., Pickrell, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+and+receptor-dependent+mitophagy+pathways+and+their+implication+in+neurodegeneration.">Ubiquitin and receptor-dependent mitophagy pathways and their implication in neurodegeneration.</a> Journal of Molecular Biology. 432 (8), 2510-2524 (2020).</li><li>Narendra, D., Tanaka, A., Suen, D. F., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+is+recruited+selectively+to+impaired+mitochondria+and+promotes+their+autophagy.">Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.</a> Journal of Cell Biology. 183 (5), 795-803 (2008).</li><li>Lazarou, M., Jin, S. M., Kane, L. A., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+PINK1+binding+to+the+TOM+complex+and+alternate+intracellular+membranes+in+recruitment+and+activation+of+the+E3+ligase+Parkin.">Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin.</a> Developmental Cell. 22 (2), 320-333 (2012).</li><li>Chen, M., 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=Mitophagy+receptor+FUNDC1+regulates+mitochondrial+dynamics+and+mitophagy.">Mitophagy receptor FUNDC1 regulates mitochondrial dynamics and mitophagy.</a> Autophagy. 12 (4), 689-702 (2016).</li><li>Ding, W. X., Yin, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy:+mechanisms,+pathophysiological+roles,+and+analysis.">Mitophagy: mechanisms, pathophysiological roles, and analysis.</a> Biological Chemistry. 393 (7), 547-564 (2012).</li><li>Parzych, K. R., Klionsky, 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=An+overview+of+autophagy:+morphology,+mechanism,+and+regulation.">An overview of autophagy: morphology, mechanism, and regulation.</a> Antioxidants and Redox Signaling. 20 (3), 460-473 (2014).</li><li>Hasegawa, 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=Autophagosome-lysosome+fusion+in+neurons+requires+INPP5E,+a+protein+associated+with+Joubert+syndrome.">Autophagosome-lysosome fusion in neurons requires INPP5E, a protein associated with Joubert syndrome.</a> The EMBO Journal. 35 (17), 1853-1867 (2016).</li><li>Presley, A. D., Fuller, K. M., Arriaga, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MitoTracker+Green+labeling+of+mitochondrial+proteins+and+their+subsequent+analysis+by+capillary+electrophoresis+with+laser-induced+fluorescence+detection.">MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection.</a> Journal of Chromatography B. Analytical Technologies in the Biomedical and Life Sciences. 793 (1), 141-150 (2003).</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+lysosomes+in+live+cells+with+LysoTracker.">Labeling lysosomes in live cells with LysoTracker.</a> Cold Spring Harbor Protocols. 2011 (2), 5571 (2011).</li><li>Pickles, S., Vigie, P., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+and+quality+control+mechanisms+in+mitochondrial+maintenance.">Mitophagy and quality control mechanisms in mitochondrial maintenance.</a> Current Biology. 28 (4), 170-185 (2018).</li><li>Ashrafi, G., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathways+of+mitophagy+for+quality+control+and+clearance+of+mitochondria.">The pathways of mitophagy for quality control and clearance of mitochondria.</a> Cell Death and Differentiation. 20 (1), 31-42 (2013).</li><li>Berezhnov, A. V., 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=Intracellular+pH+modulates+autophagy+and+mitophagy.">Intracellular pH modulates autophagy and mitophagy.</a> Journal of Biological Chemistry. 291 (16), 8701-8708 (2016).</li><li>Takemori, 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=Visualization+of+mitophagy+using+LysoKK,+a+7-nitro-2,1,3-benzoxadiazole-(arylpropyl)benzylamine+derivative.">Visualization of mitophagy using LysoKK, a 7-nitro-2,1,3-benzoxadiazole-(arylpropyl)benzylamine derivative.</a> Mitochondrion. 62, 176-180 (2022).</li><li>Maeda, M., 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=The+new+live+imagers+MitoMM1/2+for+mitochondrial+visualization.">The new live imagers MitoMM1/2 for mitochondrial visualization.</a> Biochemical and Biophysical Research Communications. 562, 50-54 (2021).</li><li>Feng, X. R., Yin, W., Wang, J. L., Feng, L., Kang, 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=Mitophagy+promotes+the+stemness+of+bone+marrow-derived+mesenchymal+stem+cells.">Mitophagy promotes the stemness of bone marrow-derived mesenchymal stem cells.</a> Experimental Biology and Medicine. 246 (1), 97-105 (2021).</li><li>Chu, C. T., 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=Cardiolipin+externalization+to+the+outer+mitochondrial+membrane+acts+as+an+elimination+signal+for+mitophagy+in+neuronal+cells.">Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells.</a> Nature Cell Biology. 15 (10), 1197-1205 (2013).</li><li>Sentelle, R. 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The protocol described here provides a method for evaluating and monitoring the dynamic process of mitophagy in living cells, involving autophagosomes, lysosomes, and mitochondrial fission, through co-staining with cell-permeant mitochondria and lysosome dyes. The method can also be used to identify mitochondria and assess mitochondrial morphology. Both dyes used in this study should be protected from light, multiple freeze-thaw cycles should be avoided, and the dyes should be stored in single-use aliquots as much as pos...

Mitochondria are the powerhouses of nearly all eukaryotic cells1,2. In addition to ATP production through oxidative phosphorylation, mitochondria play a vital role in other processes such as bioenergetics, calcium homeostasis, free radical generation, apoptosis, and cellular homeostasis3,4,5. As mitochondria generate reactive oxygen species (ROS) from multiple complexes in the electron transport chain, they are constantly stimulated by potential oxidative stress, which can eventually lead to structural damage and dysfunction when the antioxidant defense system collapses6,7. Mitochondrial dysfunction has been found to contribute to many diseases, including metabolic disorders, neurodegeneration, and cardiovascular disease8. Therefore, it is crucial to maintain healthy mitochondrial populations and their proper function. Mitochondria are highly plastic and dynamic organelles; their morphology and function are controlled by mitochondrial quality control mechanisms, including post-translational modifications (PTM) of mitochondrial proteins, mitochondrial biogenesis, fusion, fission, and mitophagy9,10. Mitochondrial fission mediated by dynamin-related protein 1 (DRP1), a GTPase of the dynamin superfamily of proteins, results in small and round mitochondria and isolates the dysfunctional mitochondria, which can be cleared and degraded by mitophagy11,12.
Mitophagy is a cellular process that selectively degrades mitochondria by autophagy, usually occurring in damaged mitochondria following injury, aging, or stress. Subsequently, these mitochondria are delivered to lysosomes for degradation10. Thus, mitophagy is a catabolic process that helps maintain the quantity and quality of mitochondria in a healthy state in a wide range of cell types. It plays a crucial role in the restoration of cellular homeostasis under normal physiological and stress conditions13,14. Cells are characterized by a complex mitophagy mechanism, which is induced by different signals of cellular stress and developmental changes. Mitophagy regulatory pathways are classified as ubiquitin-dependent or receptor-dependent15,16; the ubiquitin-dependent autophagy is mediated by the kinase PINK1 and the recruitment of ubiquitin ligase Parkin E3 to the mitochondria17,18, while receptor-dependent autophagy involves the binding of autophagy receptors to the microtubule-associated protein light chain LC3 that mediates mitophagy in response to mitochondrial damage19.
Transmission electron microscopy (TEM) is the most commonly used method, and still one of the best methods, to observe and detect mitophagy20. The morphological features of mitophagy are autophagosomes or autolysosomes formed by the fusion of autophagosomes with lysosomes, which can be observed from electron microscopy images21. The weakness of electron microscopy (EM), however, is the inability to monitor the dynamic processes of mitophagy, such as mitochondrial depolarization, mitochondrial fission, and fusion of autophagosomes and lysosomes, in the living cell20. Thus, evaluating mitophagy through imaging living organelles is an attractive alternative method for mitochondrial research. The live cell imaging technique described here uses two fluorescent dyes to stain mitochondria and lysosomes. When mitophagy occurs, damaged or superfluous mitochondria engulfed by autophagosomes are stained green by the mitochondrial dye, while the red dye stains the lysosomes. The fusion of these autophagosomes and lysosomes, referred to as autolysosomes, causes the green and red fluorescence to overlap and manifest as yellow dots, thus indicating the occurrence of mitophagy22. The cell-permeant mitochondria dye (MitoTracker Green) contains a mildly thiol-reactive chloromethyl moiety to label mitochondria23. To label mitochondria, cells are simply incubated with the dye, which diffuses passively across the plasma membrane and accumulates in active mitochondria. This mitochondria dye can easily stain live cells, and is less effective in staining aldehyde-fixed or dead cells. The lysosome dye (LysoTracker Red) is a fluorescent acidotropic probe used for labeling and tracking acidic organelles in live cells. This dye exhibits a high selectivity for acidic organelles and can effectively label live cells at nanomolar concentrations24.
The procedures for using these fluorescent dyes in living cells, including loading the dyes and the visualization of mitochondria and lysosomes, are presented here. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. It can also be used to quantify mitochondria and lysosomes, and assess mitochondrial morphology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automated cell counter</td>
			<td>Countstar</td>
			<td>IC1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>Countstar</td>
			<td>12-0005-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium (DMEM)</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Corning</td>
			<td>21-031-CVC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom cell culture dish (confocal dish)</td>
			<td>NEST</td>
			<td>801002</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J (Rasband, NIH)</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>Krebs&#8211;Henseleit(KHB) buffer</td>
			<td></td>
			<td></td>
			<td>Self-prepared</td>
		</tr>
		<tr>
			<td>LysoTracker Red</td>
			<td>Invitrogen</td>
			<td>1818430</td>
			<td>100 &#181;mol/L, red-fluorescent lysosome dye</td>
		</tr>
		<tr>
			<td>MitoTracker Green</td>
			<td>Invitrogen</td>
			<td>1842298</td>
			<td>200 &#181;mol/L stock, green-fluorescent mitochondria dye</td>
		</tr>
		<tr>
			<td>Mouse Embryonic Fibroblasts</td>
			<td></td>
			<td></td>
			<td>Self-prepared</td>
		</tr>
		<tr>
			<td>Objective (63x oil lens)</td>
			<td>ZEISS</td>
			<td>ZEISS LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Gibico</td>
			<td>Cat# 25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEISS LSM 880 Confocal Laser Scanning Microscope</td>
			<td>ZEISS</td>
			<td>ZEISS LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Microscopy Software 2.1 (confocal microscope imaging software)</td>
			<td>ZEISS</td>
			<td>ZEN 2.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing mitophagy with fluorescent dyes for mitochondria and lysosome

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

MitoTracker Green is a green-fluorescent mitochondrial stain that is able to accurately localize to mitochondria. The dye can easily stain live cells and is less effective in staining aldehyde-fixed or dead cells (Figure 2). The red-fluorescent lysosome dye LysoTracker Red is capable of labeling and tracking acidic lysosomal organelles and can only stain live cells (Figure 2). Confocal microscope imaging allows the visualization of mitochondria and lysosomes sta...

Watch this Scientific Journal Video about Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome at JoVE.com

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. ...

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