We thank Dr. Atsushi Miyawaki and Dr. Richard J. Youle for sharing the mt-Keima plasmid and mt-Keima integrated Hela cells. We thank Raghavendra A. Shamanna and Dr. Deborah L. Croteau for the critical reading of the manuscript. This research was supported by the Intramural Research Program of the NIH (VAB), National Institute on Ageing, as well as a 2014-2015 and a 2016-2017 NIA intra-laboratory grant (EFF, VAB). EFF was supported by HELSE SOR-OST RHF (Project No: 2017056) and the Research Council of Norway (Project No: 262175).
AUTHOR CONTRIBUTIONS:
EFF designed the manuscript and prepared the draft; KP, NS, EMF, RDS, JSK, SAC, YH, and ED wrote different sections of the paper; NT, JP, HN, and VAB revised the manuscript and provided expertise.

Animals (male and female mice) were born and bred in an accredited animal facility, in accordance and approval of the NIH Animal Care and Use Committee. Euthanasia methods must be consistent with all national and institutional regulations.
1. Detection of Mitophagy in Human Cells
<ol>
	<li>Detection of mitophagy using mt-Keima plasmid 
	NOTE: The Keima protein, fused to a mitochondria target signal, simplified as mt-Keima, has proved useful for in vivo mitophag...

<ol>
	<li>Fang, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+DNA+damage+signalling+to+mitochondria+in+ageing.">Nuclear DNA damage signalling to mitochondria in ageing.</a> Nat Rev Mol Cell Biol. 17 (5), 308-321 (2016).</li><li>Scheibye-Knudsen, M., Fang, E. F., Croteau, D. L., Wilson, D. M., Bohr, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+the+mitochondrial+powerhouse.">Protecting the mitochondrial powerhouse.</a> Trends Cell Biol. 25 (3), 158-170 (2015).</li><li>Sun, N., Youle, R. J., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mitochondrial+Basis+of+Aging.">The Mitochondrial Basis of Aging.</a> Mol Cell. 61 (5), 654-666 (2016).</li><li>Shadel, G. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+mitophagy+in+XPA+via+PARP-1+hyperactivation+and+NAD(+)/SIRT1+reduction.">Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction.</a> Cell. 157 (4), 882-896 (2014).</li><li>Diot, A., 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=A+novel+quantitative+assay+of+mitophagy:+Combining+high+content+fluorescence+microscopy+and+mitochondrial+DNA+load+to+quantify+mitophagy+and+identify+novel+pharmacological+tools+against+pathogenic+heteroplasmic+mtDNA.">A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.</a> Pharmacol Res. 100, 24-35 (2015).</li><li>Schiavi, A., 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=Iron-Starvation-Induced+Mitophagy+Mediates+Lifespan+Extension+upon+Mitochondrial+Stress+in+C.+elegans.">Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans.</a> Curr Biol. 25 (14), 1810-1822 (2015).</li><li>Rosado, C. J., Mijaljica, D., Hatzinisiriou, I., Prescott, M., Devenish, 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=Rosella:+a+fluorescent+pH-biosensor+for+reporting+vacuolar+turnover+of+cytosol+and+organelles+in+yeast.">Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast.</a> Autophagy. 4 (2), 205-213 (2008).</li><li>Sargsyan, A., 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=Rapid+parallel+measurements+of+macroautophagy+and+mitophagy+in+mammalian+cells+using+a+single+fluorescent+biosensor.">Rapid parallel measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor.</a> Sci Rep. 5, 12397 (2015).</li><li>Altun, Z. F., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+C.+elegans+anatomy.">Introduction to C. elegans anatomy.</a> WormAtlas. , (2009).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+worm+lab:+from+culturing+worms+to+mutagenesis.">An introduction to worm lab: from culturing worms to mutagenesis.</a> J Vis Exp. (47), e2293 (2011).</li><li>Palikaras, K., 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=In+vivo+Mitophagy+Monitoring+in+Caenorhabditis+elegans+to+Determine+Mitochondrial+Homeostasis.">In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis.</a> Bio Protoc. 7 (7), e2215 (2017).</li><li>Palikaras, K., 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=Assessing+Mitochondrial+Selective+Autophagy+in+the+Nematode+Caenorhabditis+elegans.">Assessing Mitochondrial Selective Autophagy in the Nematode Caenorhabditis elegans.</a> Methods Mol Biol. 1567, 349-361 (2017).</li><li>Sun, N., 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=A+fluorescence-based+imaging+method+to+measure+in+vitro+and+in+vivo+mitophagy+using+mt-Keima.">A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.</a> Nat Protoc. 12 (8), 1576-1587 (2017).</li><li>Eiyama, A., 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=Assays+for+Mitophagy+in+Yeast.">Assays for Mitophagy in Yeast.</a> Methods Mol Biol. 1567, 337-347 (2017).</li><li>McWilliams, T. G., 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=mito-QC+illuminates+mitophagy+and+mitochondrial+architecture+in+vivo.">mito-QC illuminates mitophagy and mitochondrial architecture in vivo.</a> J Cell Biol. 214 (3), 333-345 (2016).</li></ol>

Accurate measurement of mitophagy is technically demanding. Here, we have presented several robust techniques which allow for both qualitative detection of mitophagy and quantification of mitophagy levels in the most common laboratory experimental models.
To acquire replicable data, an experimental design with at least three biological repeats is necessary. All researchers involved in experimentation and analysis must be blinded to experimental group identities. Furthermore, imaging fields mus...

Mitophagy is essential for mitochondrial maintenance. Mitochondria intersect multiple cell signaling pathways and are universal sub-cellular organelles responsible for cellular energy production, cell metabolism, and calcium homeostasis1,2,3,4. Mitochondria constantly experience challenges from endogenous and exogenous sources, such as reactive oxygen species (ROS) and mitochondrial toxicants, respectively, which lead to the generation of &#34;aged&#34; and dysfunctional mitochondria. Accumulation of damaged mitochondria decreases the efficiency of ATP production while increasing the amount of harmful ROS, and has been linked to age-related diseases such as metabolic diseases, AD, and PD1,5,6. To prevent mitochondria induced cellular dysfunction, cells need to specifically recognize damaged mitochondria and efficiently remove them through a cellular process termed mitochondrial autophagy (mitophagy). This demonstrated importance of mitophagy in health and disease illustrates the need for accurate and efficient methods to detect mitophagy both in vitro and in vivo.
Mitophagy is a multiple-step process involving many proteins and protein complexes5,7,8. In brief, a damaged mitochondrion is first recognized and engulfed by a double-membraned phagophore, which can originate from the plasma membrane, endoplasmic reticulum, Golgi complex, nucleus, or mitochondrion itself9,10. The spherical phagophore elongates and eventually seals the mitochondria inside, constituting the mitochondrial autophagosome (mitophagosome). The mitophagosome then fuses with the lysosome for degradation, forming an autolysosome in which the damaged mitochondrion is degraded and recycled7,8. Major autophagic proteins also involved in mitophagy include: Autophagy Related 7 (ATG7) and Beclin1 (initiation), Microtubule-Associated Protein 1A/1B-Light Chain 3 (LC3-II) (LGG-1 in C. elegans) and p62 (components of phagophore), and lysosomal-associated membrane glycoprotein 2 (LAMP2)6,7. In addition, there are several essential proteins unique to mitophagy, including PTEN-induced Putative Kinase 1 (PINK-1), Parkin1, Nuclear Dot Protein 52 kDa (NDP52), optineurin, BCL2 Interacting Protein 3 Like (NIX/BNIP3L) (DCT-1 in C. elegans), among others5,6,11.
A common method to detect changes in levels of autophagy is by the ratio of LC3-II/LC3-I or LC3-II/actin. However, this method is nonspecific, as an increase in this ratio may reflect either an increased initiation or an impaired fusion of mitophagosome to lysosome12. Another method is to evaluate the colocalization between an autophagy marker (e.g., LC3) and a mitochondrial protein (e.g., Translocase of Outer Mitochondrial Membrane 20 (TOMM20, which could be degraded by proteasomes)). However, this can only indicate changes in total mitophagy levels and cannot distinguish the step(s) at which blockage occurs. This can be clarified by using lysosomal inhibitors (e.g., E64d+Pepstatin A, termed EP) in parallel to cause the accumulation of mitophagosomes. The difference between the number of mitophagosomes at baseline and the number of mitophagosomes following treatment with EP can indicate mitophagy. These limitations have prompted the development of novel mitophagy detection techniques. In view of the increasing relevance of mitophagy in a wide spectrum of human diseases, we present several robust mitophagy detection techniques which may be useful for researchers. We cover both in vitro and in vivo techniques and recommend combining multiple techniques to verify changes of mitophagy.

<table>
	<tbody>
		<tr>
			<td>mt-Keima mouse</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 DNA transfection reagent</td>
			<td>Thermofisher</td>
			<td>#11668027</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM medium (Gibco)</td>
			<td>Thermofisher</td>
			<td>#31985062</td>
			<td>serum-free medium</td>
		</tr>
		<tr>
			<td>mtKemia plasmid: pCHAC-mt-mKeima</td>
			<td>addgene</td>
			<td>#72342</td>
			<td></td>
		</tr>
		<tr>
			<td>COXII antibody (mouse)</td>
			<td>abcam</td>
			<td>#ab110258</td>
			<td></td>
		</tr>
		<tr>
			<td>LAMP2 antibody (rabbit)</td>
			<td>NOVUS</td>
			<td>#CD107b</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-anti-rabbit with wavelength 568 nm of red fluorescent protein (RFP)</td>
			<td>Thermofisher</td>
			<td>#Z25306</td>
			<td>Alexa Fluor 568 dye</td>
		</tr>
		<tr>
			<td>goat-anti-mouse with wavelength 488 nm of green fluorescent protein (GFP)</td>
			<td>Thermofisher</td>
			<td>#Z25002</td>
			<td>Alexa Fluor 488 dye</td>
		</tr>
		<tr>
			<td>prolong gold antifade with DAPI</td>
			<td>Invitrogen</td>
			<td>#P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>SIGMA 
			Corning Costar</td>
			<td>#CLS3516</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well chamber slide</td>
			<td>THermofisher, Nunc Lab-Tek</td>
			<td>#171080</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc F 96-well plate</td>
			<td>Thermofisher</td>
			<td>#152038</td>
			<td></td>
		</tr>
		<tr>
			<td>LC3B antibody rabbit</td>
			<td>NOVUS</td>
			<td>#NB100-2220</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA antibody</td>
			<td>Progen Biotechnik</td>
			<td>#anti-DNA mouse monoclonal, AC-30-10</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermofisher</td>
			<td>#D1306</td>
			<td>antifade mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>IN Cell analyzer (fluorescent reader )</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#IN Cell analyzer 2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse TE-2000e confocal microscope</td>
			<td>Nikon</td>
			<td>#TE-2000e</td>
			<td></td>
		</tr>
		<tr>
			<td>Colocalization software</td>
			<td>Volocity</td>
			<td>#Volocity 6.3</td>
			<td>alternative Zeiss ZEN 2012 software</td>
		</tr>
		<tr>
			<td>IN Cell Investigator Software</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#28408974</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture medium</td>
			<td>Thermofisher</td>
			<td>#DMEM&#8211;Dulbecco&#39;s Modified Eagle Medium</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermofisher</td>
			<td>#15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>#12003C-1000ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture Incubator</td>
			<td>Thermofisher</td>
			<td>#Thermo Forma 3110 CO2 Water Jacketed Incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>epifluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axio Imager Z2</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Olympus</td>
			<td></td>
			<td>Olympus DP71</td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axio Observer Z1</td>
		</tr>
		<tr>
			<td>confocal software</td>
			<td>Zeiss</td>
			<td></td>
			<td>ZEN 2012</td>
		</tr>
		<tr>
			<td>image analysis software</td>
			<td>Image J</td>
			<td>colocalization analysis, etc</td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>statistical analysis software</td>
			<td>GraphPad Software Inc., San Diego, USA</td>
			<td></td>
			<td>GraphPad Prism software package</td>
		</tr>
		<tr>
			<td>material to make a worm pick</td>
			<td>Surepure Chemetals</td>
			<td>#4655</td>
			<td>The pick is made of 30 gauge 90% platinum 10% iridium wire</td>
		</tr>
		<tr>
			<td>IR: N2;Ex[pmyo-3 TOMM-20::Rosella]</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td>Maintain transgenic animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>IR: N2; Ex[pdct-1 DCT-1::GFP; pmyo-3 DsRed::LGG-1]</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pmyo-3 TOMM-20::Rosella</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pdct-1 DCT-1::GFP</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pmyo-3 DsRed::LGG-1</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraquat solution</td>
			<td>see supplementary data for preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M9 buffer</td>
			<td>see supplementary data for preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M9-levamisole buffer</td>
			<td>see supplementary data for preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Microscope Slides and Coverslips</td>
			<td>Fisher Scientific</td>
			<td>#B9992000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>STERIS Animal Health</td>
			<td>19 Piece Canine Spay Pack Economy</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>STERIS Animal Health</td>
			<td>19 Piece Canine Spay Pack Economy</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Thermofisher</td>
			<td>#AM9625</td>
			<td>10x PBS needs to be diluted to 1x PBS by using ddH2O</td>
		</tr>
		<tr>
			<td>shaker</td>
			<td>Fisher Scientific</td>
			<td>#11-676-178</td>
			<td>Thermo Scientific MaxQ HP Tabletop Orbital Small Open Air Platform Shaker Package A</td>
		</tr>
		<tr>
			<td>2% agarose pad</td>
			<td>see supplementary data for preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vibroslice blades</td>
			<td>World precision instruments</td>
			<td>#BLADES-2</td>
			<td>single-edge blade</td>
		</tr>
		<tr>
			<td>metal plate</td>
			<td>MSC</td>
			<td>#78803988</td>
			<td>0.012 in thick x 6 in wide x 12 in long, 430 Stainless Steel Sheet</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>Methyl viologen dichloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>#856177</td>
			<td>paraquat</td>
		</tr>
		<tr>
			<td>Incubator for nematodes</td>
			<td>AQUALYTIC</td>
			<td></td>
			<td>Incubator to maintain 20 &#176;C</td>
		</tr>
		<tr>
			<td>Dissecting stereomicroscope</td>
			<td>Olympus</td>
			<td>SMZ645</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver Z1</td>
			<td>For nematodes (step 2)</td>
		</tr>
		<tr>
			<td>epifluorescence microscope</td>
			<td>Zeiss</td>
			<td>AxioImager Z2</td>
			<td>For nematodes (step 2)</td>
		</tr>
		<tr>
			<td>UV crosslinker</td>
			<td>Vilber Lourmat</td>
			<td>BIO-LINK &#8211; BLX-E365</td>
			<td>UV light source; 356 nm</td>
		</tr>
	</tbody>
</table>

in vitro and in vivo detection of mitophagy in human cells, c. elegans, and mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

Detection of Mitophagy in Human Cells:
Using the procedure presented here, human HeLa cells were transfected with mt-Keima plasmid. Healthy cells demonstrated a well-organized mitochondrial network (GFP, 488 nm) with few incidences of mitophagy (RFP, 561 nm). However, cells pretreated with a mitochondrial uncoupler FCCP (30 &#181;M for 3 h) exhibited a profound increase in mitophagy incidence (Figure 1A</strong...

Watch this Scientific Journal Video about In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice at JoVE.com

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

The overall goal of these experiments is to evaluate levels of mitophagy in human cells, C.elegans, and mice. Mitophagy has become a very important concept in the crossroads of neuro-degeneration, metabolic diseases and aging, and insight into the methodologies that are presented here and future use of these could be helping to make advances in this important field. The main advantages of this technique are that it detects mitophagy in a robust and efficient manner and enables the detection of mitophagy both in vitro and in vivo.This technique presented here will assist researchers to develop new insight and to develop new strategies for therapy against neuro-generative diseases, including Alzheimer's and Parkinson's, where we know there is a mitochondrial dysfunction. In addition to providing insights into the molecular mechanisms of mitophagy, these method may lead to the discovery of novel drug candidates through the screening of mitophage inducers. Visual demonstrations of these methods is critical as levels of mitophagy are very sensitive to changes in experimental conditions in both C.elegans and mice.Seed 300, 000 to one million cells in a 35 millimeter glass bottom microwell dish, 10 millimeter microwell, so that the cells reach 70%confluency on the next day. Maintain the cells in complete cell culture medium and incubate them in an incubator at 37 degrees Celsius and 5%carbon dioxide. The next day, dilute 15 microliters of the DNA transfection reagent with 150 microliters of serum free medium according to the manufacturer's protocol.Also, dilute two to five micrograms of the mito-Keima plasmid DNA with 150 microliters of serum free medium. Add the diluted mito-Keima plasmid to the diluted DNA transfection reagent and vortex the mixture for 10 seconds. Then incubate the mixture for 10 minutes at room temperature.Next, add the DNA transfection reagent mixture drop-wise to the cells, gently shake the plates for five to 10 seconds to mix the solution. At this point, place the cells back into the incubator and incubate them for 24 hours. The next day, change the media to fresh complete cell culture medium and then incubate the transfected cells for an additional 24 hours.Image the transfected cells using confocal microscopy. Randomly image 20 areas under 40 times magnification in order to document a total of 100 to 200 cells in every well. On day one, pick L4 larvae of transgenic animals expressing both DCT-1 GFP and DSRed LGG-1 in body wall muscle cells and place them onto an OP50 seeded nematode growth media plate.Place five to 10 worms onto each 3.5 centimeter plate using at least three plates. When finished, incubate the nematodes at 20 degrees Celsius. Next, on day five, synchronize the nematodes by selecting 15 to 20 L4 transgenic larvae and transferring them onto fresh OP50 seeded plates.Use at least five plates for each experimental condition. On day seven, prepare some of the vehicle plates for mitophagy affecting drugs of interest and some to use as positive controls. Next, expose E.coli seeded plates to UV light for 15 minutes at an intensity of 222 microwatts per centimeter squared, to ensure the mitophagy inducing compounds on the plates are not metabolized by bacteria.Now, add the compound of interest in 10 microliters to the top of the seeded plates and add an equivalent amount of compound vehicle to the vehicle plates as a negative control. For positive control of mitophagy induction, add paraquat, a mitochondrial toxicant. Gently swirl the plates until the solution coats the entire surface, and allow the plates to dry with the lids closed, at room temperature, for at least one hour.Once dry, transfer 10 to 20 two-day old adult transgenic animals to the plates. Incubate them at 20 degrees Celsius for two days. On day nine, prepare 2%agarose pads and add one droplet of 20 millimolar levamisole in M9, per pad.Immobilize the transgenic animals for imaging by placing them in the M9 levamisole drop. Then, gently place a cover slip on the top of the drop. Place the sample on a confocal microscope stage and image the single body wall muscle cells by taking Z-stack images under 63 times magnification.Place a fresh, isolated mito-Keima mouse liver on a chilled metal plate, on ice, to rapidly cool the tissue. Keep it there while processing the tissue as quickly as possible, as signal from the mito-Keima protein remains stable on ice for up to one hour. Next, use a pair of curved forceps to lift the liver sample and rinse it with five milliliters of ice cold PBS.Finally, image the tissue sections using confocal microscopy with two sequential excitations. Using the procedure presented here, human HeLa cells were transfected with the mito-Keima plasmid. Healthy cells demonstrated a well-organized mitochondrial network with few incidences of mitophagy.However, cells pretreated with a mitochondrial uncoupler FCCP, exhibited a profound increase in mitophagy incidence. Mitophagy was also measured using the co-localization of LAMP2 and COXII. For in vivo detection of mitophagy in C.elegans, transgenic nematodes expressing MT rosella in body wall muscle cells were treated with eight millimolars of paraquat.The level of mitophagy induction indicated by a decrease in the pH-sensitive fluorescence ratio, was found to be significantly decreased in the nematodes treated with paraquat. Additionally, the elevated number of co-localization events between DCT-1 fused GPF and DSRed fused LGG-1, signifies mitophagy stimulation in response to oxidative stress. Finally, these images represent mito-Keima signals from the liver and cerebellum of a mito-Keima mouse.Tissues need to be kept cold and imaged as quickly as possible, but when prepared properly, can be used to provide insight into mitophagy in normal and pathophysiological conditions. The use of this technique has paved the ways for researchers in the broad field of neuro-degeneration and mitochondria biology, to explore mitochondrial functions in different model systems including cultured cells, worms, or C.elegans, and mouse models. After watching this video, you should have a grasp of multiple methods to use in the quantitative evaluation of mitophagy levels in both C.elegans and mice.

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17.2K 조회수.  National Institutes of Health. The overall goal of these experiments is to evaluate levels of mitophagy in human cells, C.elegans, and mice. Mitophagy has become a very important concept in the crossroads of neuro-degeneration, metabolic diseases and aging, and insight into the methodologies that are presented here and future use of these could be helping to make advances in this important field. The main advantages of this technique are that it detects mitophagy in a robust and efficient manner and enables the detection o...