This work was supported by a grant from National Research Foundation of Korea (2016R1D1A1B03931949) (to J. U.), and by the National Research foundation of Korea (NRF) grant funded by the Korea government(MSIT) (No. 2016R1A2B2008887, No. 2016R1A5A2007009) (to J.Y.)

1. Generation of HeLa cells expressing mito-Keima (mt-Keima)
<ol>
	<li>Preparation of mt-Keima lentivirus
	<ol>
		<li>Coat a 100-mm culture dish by adding 2 mL of 0.001% poly-L-lysine/phosphate-buffered saline (PBS) and allow it to stand for 5 min at room temperature.</li>
		<li>Remove the poly-L-lysine solution using a glass pipette connected to a vacuum and wash the culture dish by adding 2 mL of 1x PBS.</li>
		<li>Plate 1.5 x 106 HEK293T cells on the coated culture dish with 10 mL of DMEM...

<ol>
	<li>McBride, H. M., Neuspiel, M., Wasiak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+More+than+just+a+powerhouse.">Mitochondria: More than just a powerhouse.</a> Current Biology. 16 (14), R551-R560 (2006).</li><li>Zorov, D. B., Krasnikov, B. F., Kuzminova, A. E., Vysokikh, M., Zorova, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+revisited.+Alternative+functions+of+mitochondria.+Bioscience+Reports.">Mitochondria revisited. Alternative functions of mitochondria. Bioscience Reports.</a> Bioscience Reports. 17 (6), 507-520 (1997).</li><li>Taylor, R. W., Turnbull, D. 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E., 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=Drosophila+pink1+is+required+for+mitochondrial+function+and+interacts+genetically+with+parkin.">Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin.</a> Nature. 441 (7097), 1162-1166 (2006).</li><li>Park, 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=Mitochondrial+dysfunction+in+Drosophila+PINK1+mutants+is+complemented+by+parkin.">Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin.</a> Nature. 441 (7097), 1157-1161 (2006).</li><li>Narendra, D., Tanaka, A., Suen, D. F., Youle, R. 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F., Toth, R., James, J., Ganley, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+iron+triggers+PINK1/Parkin-independent+mitophagy.">Loss of iron triggers PINK1/Parkin-independent mitophagy.</a> EMBO Reports. 14 (12), 1127-1135 (2013).</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>Kubli, D. 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Here, we present a rapid and sensitive method for using flow cytometry to measure cellular mitophagy activity in cells expressing mt-Keima. Cells undergoing a high level of mitophagy exhibit an increased ratio of PE-CF594 (561 nm)/BV605 (405 nm) excitation. Thus, mitophagy activity can be expressed as the percentage of cells exhibiting a high 561/405 ratio. We calculated the percentage of cells in the &#34;high&#34; gate region on a dot plot of PE-CF594 (561 nm) versus BV605 (405 nm), and the results showed that treatmen...

Mitochondria are organelles that are essential for cell proliferation and physiology. Mitochondria are responsible for generating more than 80% of the ATP supply via oxidative phosphorylation, and they also provide various metabolic intermediates for biosynthesis and metabolism1,2. In addition to their roles in energy supply and metabolism, mitochondria play central roles in many other important processes, including reactive oxygen species (ROS) generation, the regulation of cell death, and intracellular Ca2+ dynamics3. Alterations in mitochondrial function have been associated with human diseases, including cancer, diabetes mellitus and various neurodegenerative diseases4,5. Increased mitochondrial dysfunction and mitochondrial DNA mutations are also thought to contribute to normal aging processes6,7,8. Therefore, quality control is a crucial issue in mitochondria. Mitochondria are highly dynamic organelles that can continuously change their shape between elongated networks and short, fragmented form. Mitochondria dynamics plays an important role in maintaining the function of mitochondria as well as their degradation through mitophagy9.
Mitophagy is a mechanism that involves the selective degradation of whole mitochondria using the autophagy machinery. Mitophagy is the principle mechanism underlying mitochondrial turnover and the removal of damaged or dysfunctional mitochondria. In this process, mitochondria are first surrounded by a membrane, resulting in the formation of autophagosomes, which then fuse with lysosomes for hydrolytic degradation9. Previous genetic studies in Drosophila have suggested that two Parkinson&#39;s disease-related genes, PTEN-induced putative kinase 1 (PINK1) (PARK6) and Parkin (PARK2), function in the same pathway10,11. Subsequence studies have shown that the PINK1-Parkin pathway is responsible for eliminating damaged mitochondria and defects in this pathway result in dysfunctional mitophagy and may contribute to human diseases12,13. Defects in mitophagy processes have recently been found in various human diseases, including cancer, heart disease, liver diseases and neurodegenerative disease 14. In addition, recent studies have also shown that mitophagy is critical to many physiological processes, such as differentiation, development, and the immune response15,16,17,18,19, suggesting that mitophagy may play a more active role in controlling cell functions.
Despite recent confirmation that mitophagy plays an important role in both quality control in mitochondria and human disease, the molecular mechanisms underlying mitophagy remain poorly understood. Although mitophagy-related mechanisms have been systematically studied in yeast, studies aimed at exploring mitophagy-related mechanisms in mammalian cells have mainly focused on the PINK1-Parkin pathway. Previous studies have established that the PINK1-Parkin pathway is primarily responsible for the selective removal of damaged mitochondria via mitophagy12,20,21. However, recent studies have reported that in some models, mitophagy can be activated even in the absence of functional PINK122,23,24. These results suggest that in addition to the PINK1-Parkin pathway, there an additional unidentified pathway that can activate mitophagy.
The absence of a convenient method to assess mitophagy activity is a major obstacle to exploring the pathways that regulate mitophagy and its role in pathophysiological events. Electron microscopy is a powerful tool to directly visualize autophagosome-engulfed mitochondria. However, electron microscopy has limitations in quantifying mitophagy activity. Although strategies that use microtubule-associated protein-1 light chain 3 (LC3)-conjugated fluorescent probes, such as GFP-LC3, are currently the most widely used approaches25, the transient nature of the LC3-based signal and its high rate of false-positive signals limit its sensitivity for detecting mitophagy in cells26. A combination of western blotting to measure mitochondrial protein level, the quantification of mitochondrial DNA, and fluorescence microscopy analysis to colocalize mitochondria with either autophagosomes or lysosomes would be a good approach for assessing mitophagy. However, quantification limitations and a lack of convenience of existing methodologies call for new approaches. The introduction of a new pH-dependent fluorescent protein, the mitochondrial target Keima (mt-Keima), greatly improved the ability to detect mitophagy27. By fusing the mitochondrial-targeting sequence of cytochrome c oxidase subunit VIII (COX VIII) into Keima, mt-Keima is directed to the mitochondrial matrix. The large shift in the excitation peak of Keima from 440 nm to 586 nm (corresponding to pH 7 and pH 4, respectively) can be utilized to assess mitophagy with good sensitively in both in vitro and in vivo experiments28,29. More importantly, the resistance of mt-Keima to lysosomal degradation causes the integration of the mt-Keima signal in lysosomes, allowing for the easy quantitative measurement of mitophagy activity. The fluorescence change that occurs in mt-Keima can be analyzed using either confocal microscopy or flow cytometry28,29. However, a flow cytometry-based method for measuring mitophagy activity using mt-Keima has not been provided in detail to date.
Here, we describe a detailed protocol for a flow cytometry-based technique for measuring mitophagy activity in cells using mt-Keima. Although we have shown here that flow cytometry analysis successfully detected CCCP-induced mitophagy in HeLa cells expressing Parkin, this technique can be applied to a wide variety of cell types.

<table border="0" cellpadding="0" cellspacing="0" style="width:635px;" width="635">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">REAGENTS</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">poly-L-lysine</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
			<td style="width:133px;">wellgene</td>
			<td style="width:119px;">LS202-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:133px;">Hyclone</td>
			<td style="width:119px;">SH30013.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEK293T</td>
			<td style="width:133px;">ATCC</td>
			<td style="width:119px;">CRL-3216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">12800-082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OPTI-MEM&#160;</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Turbofect</td>
			<td style="width:133px;">Thermos scientific</td>
			<td style="width:119px;">R0531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.45 &#956;m syringe filter</td>
			<td style="width:133px;">sartorius</td>
			<td align="right" style="width:119px;">16555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HeLa</td>
			<td style="width:133px;">ATCC</td>
			<td style="width:119px;">CCL-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polybrene</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9268</td>
			<td>8 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puromycin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8833</td>
			<td>2 mg/ml&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Carbonyl cyanide m-chlorophenyl hydrazine (CCCP)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">C2759</td>
			<td>10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trypsin-EDTA</td>
			<td style="width:133px;">wellgene</td>
			<td style="width:119px;">LS015-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EQUIPMENTS</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD LSRFortessa</td>
			<td style="width:133px;">BD Bioscience</td>
			<td style="width:119px;">LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSDIVA</td>
			<td style="width:133px;">BD Bioscience</td>
			<td style="width:119px;">FACSDIVA (v8.0.1)</td>
			<td></td>
		</tr>
	</tbody>
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sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

An example of the flow cytometry analysis of CCCP-induced mitophagy in HeLa-Parkin cells is shown in Figure 4. Using the flow cytometry analysis method described above, we can detect a dramatic increase in mitophagic cells in the &#34;high&#34; gate. The percentage of cells in the &#34;high&#34; gate was increased more than 10-fold compared with untreated control cells (Figure 4A). This increase in mitophagy activity was complete...

Watch this Scientific Journal Video about Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima at JoVE.com

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

The overall goal of this procedure is to provide a sensitive technique for measuring cellular mitophagy activity using flow cytometry. Here we demonstrate that this method was successfully used to assess the dramatic increase in HeLa cell mitophagy, after treatment with CCCP, a mitochondrial uncoupler. This technique allows mitophage activity to be measured very quickly and easily.The main advantage of this technique is that you can sensitively measure mitophage activity in living cells without any additional staining. Therefore, this technique will ultimately be able to help in understanding the mechanism and physiological role of mitophage. Demonstrating the procedure will be Jee-Hyun Um and Young Yeon Kim from my laboratory.Begin this procedure by preparing the mt-Keima, a mitochondria-targeting Keima lentivirus. First seed HEK293T cells in a poly-L-lysine coated culture dish and culture them at 37 degrees Celsius in a tissue culture incubator for one day. On the next day transfect HEK293T cells with mt-Keima lentiviral DNA and packaging DNase using a transfection reagent according to the manufacturer's instructions.After eight hours of transfection remove the transfection media and add eight milliliters of fresh media. Collect the media containing viral particles 48 hours later and remove cellular debris by centrifugation. Next filter the virus-containing media with a 0.45 micrometer syringe filter.Plate HeLa-Parkin cells in a 60 millimeter culture dish one day before harvesting the mt-Keima lentivirus and culture the cells with four milliliters of growth media for one day at 37 degrees Celsius in an incubator. On the next day remove the growth medium add one millimeter of mt-Keima virus media add an additional two milliliters of growth medium containing 3 microliters of polybrene stock solution and incubate the cells for one day in a CO2 incubator. At 24 hours after infection remove the virus media wash the cells twice with PBS and add four milliliters of growth media.To remove uninfected cells treat cells for two days with two micrograms per microliter puromycin. Prepare fresh media containing 10 micromolar CCCP. Remove the cultured media and add four milliliters of fresh growth media containing CCCP.Incubate the cells at 37 degrees Celsius in a CO2 incubator for six hours. Remove the growth medium and wash the cells once with PBS. Detach the cells by treatment with trypsin EDTA solution.Collect the cells by centrifugation and re-suspend them in one milliliter of PBS. Transfer the cells in a FACS tube and place them on ice. For flow cytometry turn on the flow cytometer and start the analyzing software.Generate a new experiment and select BV605 for the violet laser and PE-CF594 for the yellow-green laser in the parameter window. First run a control sample of cells not infected with the mt-Keima lentivirus. In the plot of forward scatter versus side scatter draw a gate, using the polygon icon, around the live cells and eliminate dead cells and cell debris.Then run HeLa-Parkin cells infected with the mt-Keima lentivirus. In the dot plot of BV605 versus side scatter adjust the voltage so that HeLa-Parkin cells expressing mt-Keima are clearly distinguished from control cells that do not express mt-Keima. Next in the dot plot of PE-CF594 versus side scatter adjust the voltage so that HeLa-Parkin cells expressing mt-Keima are clearly distinguished from control cells.Then in the dot plot of BV605 versus PE-CF594 select the mt-Keima-positive cell population by drawing a gate using the polygon icon. Next create another dot plot of BV605 versus PE-CF594 showing only mt-Keima gated cells. In this dot plot, draw a gate around untreated mt-Keima-positive cells.This gate is referred to as the low gate. Then draw another gate containing the region above the low gate. This gate is referred to as the high gate because it contains cells with high mitophagy activity.Then select the Show Population Hierarchy menu. The Percent of Parent value represents the percentage of cells in the high gate among the mt-Keima-positive population. Now run each sample and record at least 10 000 events in the mt-Keima stopping gate.Flow cytometry analysis of untreated HeLa cells expressing mt-Keima showed a low mitophagy level. CCCP treatment induced a dramatic increase in mitophagy cells in the high gate. The percentage of mitophagy cells increased by more than 10 fold at six hours after CCCP treatment.This increase in mitophagy by CCCP treatment was completely abolished by co-treatment with bafilomycin A.Once the cells expressing mito-Keima are ready mitophage measurement using flow cytometry can be conducted within one hour if prepared properly. For accurate mitophage analysis by flow cytometry it is important to set up the correct high and low gates. When analyzing new types of cells it is necessary to use well-known cells such as HeLa cells as a control.In addition it is important to remember that mitophage should be fully confirmed by additional characteristics such as a decrease in mitochondrial protein and a reduction in mitochondrial DNA. This technique can be applied to a wide variety of cell types. After watching this video you should have a good understanding of how to analyze mitophage by flow cytometry.

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14.4K visualizações.  Dong-A University. The overall goal of this procedure is to provide a sensitive technique for measuring cellular mitophagy activity using flow cytometry. Here we demonstrate that this method was successfully used to assess the dramatic increase in HeLa cell mitophagy, after treatment with CCCP, a mitochondrial uncoupler. This technique allows mitophage activity to be measured very quickly and easily.The main advantage of this technique is that you can sensitively measure mitophage activity in living cell...