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. 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=Ceramide+targets+autophagosomes+to+mitochondria+and+induces+lethal+mitophagy.">Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy.</a> Nature Chemical Biology. 8 (10), 831-838 (2012).</li></ol>

The authors have no conflicts of interest to disclose.

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...

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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. ...

This method helps to observe mitophagy in living cells using a cell permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye. Mitophagy plays an important role in maintaining mitochondria homeostasis and various aspects of cellular function. This technique could provide a new approach to the treatment of mitochondria-related diseases.This procedure is not very complicated. Capturing clear images is very important for counting the overlay of mitochondria and lysosomes. To begin, culture mouse embryonic fibroblast or MEF cells in a 10 centimeter cell culture dish with 10 milliliters of Dulbecco's Modified Eagle Medium or DMEM.Incubate at 37 degrees Celsius and 5%carbon dioxide and monitor the cells under a microscope at 100X magnification. When the cells reach 80 to 90%confluency, wash them with two milliliters of Dulbecco's phosphate buffered saline. Then add two milliliters of 0.05%trypsin EDTA for one minute to dissociate the cells, followed by two milliliters of DMEM to stop the reaction.Centrifuge the cell suspension at 100 G for three minutes and resuspend the pellet in one milliliter DMEM. Count the cells using an automated cell counter and cell counting chamber slides and inoculate 1.5 times 10 to the sixth cells into a new 10 centimeter cell culture dish containing 10 milliliters of DMEM. For the mitophagy assay, prepare a cell suspension as described and dilute the cell suspension to one times 10 to the fifth cells per milliliter of fresh DMEM.Add two milliliters of the diluted cell suspension to a 20 millimeter confocal dish and shake the dish in a cross. Incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours. Prepare working solutions of the dyes by diluting the stock solutions.Add two microliters each of 0.2 millimolar mitochondrial dye and 50 micromolar lysosome dye to two milliliters of DMEM to obtain a working concentration of 0.2 micromolar mitochondrial dye and 50 nanomolar lysosome dye. Remove the medium from the confocal culture dish and add one milliliter of the staining solution to cover the cells. Place the cell culture dish at 37 degrees Celsius and 5%carbon dioxide for 20 to 30 minutes.Set the parameters of the confocal microscopy imaging software. For dual excitation images, use sequential excitation at 488 nanometers and 543 nanometers and collect emission at 505 to 545 nanometers and greater than 560 nanometers respectively. Remove the culture medium dish containing the dye from the incubator and add one milliliter of Krebs-Henseleit or KH buffer to the dish.To induce mitophagy, treat the cells with FCCP at one micromolar in KH buffer for 10 minutes at room temperature and immediately proceed to image the cells using the confocal microscope. Apply an appropriate amount of oil to the top of the 63X oil lens. Place the sample on the sample stage of the confocal microscope and move it directly above the objective lens.Use the imaging software to find the sample by clicking on the Locate tab in the top left corner of the software interface. Select a green filter set for the experiment. Use the coarse adjustment knob to quickly focus by moving the objective lens up and down.After the cell sample is clearly visible through the eyepiece, search and focus the area of single cells and move it to the center of the field of view. Click on the Acquisition tab in the top left corner of the software interface to acquire images. Select only the 488 nanometer channel and the frame resolution 1024 by 1024 for preview.Click on the Live tab in the top left corner to start a live scan. Adjust the field of view to the sharpest and adjust the laser power by moving the slider left or right. Keep the gain setting below 600 to avoid over exposure.Adjust the pinhole value to 156, gain value to 545 and digital offset value to zero. Select the best field of view, check the two channels and choose the frame resolution 1024 by 1024. Click Snap to acquire 2D images and save the acquired images.In this study, FFCP was used to trigger mitophagy in MEF cells for confocal imaging. Representative images of cells stained with the green-fluorescent mitochondria dye showing mitochondria and stained with the red-fluorescent lysosome dye showing lysosome are shown here. When damaged green-stained mitochondria are engulfed by red-stained lysosomes, the green and red fluorescence overlap to reveal yellow co-localized mitochondria lysosomes.The yellow dots correspond to these co-localized mitochondria lysosomes representing ongoing mitophagy and thus can be counted to evaluate the extent of mitophagy. Preparation of a working solution of dyes for mitochondria and lysosome staining and maintaining appropriate cell confluency when imaging with confocal microscopy are key steps to remember. This procedure can also be used to analyze mitochondrial number and morphology which is important for assessing mitochondrial dynamics.Mitophagy is closely associated with a variety of human diseases and this technique can be used to explore the connection between mitophagy and human diseases.

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JoVE Journal

Biology

3.7K Views.  Tongji University. This method helps to observe mitophagy in living cells using a cell permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye. Mitophagy plays an important role in maintaining mitochondria homeostasis and various aspects of cellular function. This technique could provide a new approach to the treatment of mitochondria-related diseases.This procedure is not very complicated. Capturing clear images is very important for counting the overlay of mitochondria and lysoso...