Name: in vitro and in vivo detection of mitophagy in human cells, c. elegans, and mice
Uploaded: 2017-11-22T13:00:00
Description: Watch this Scientific Journal Video about In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice at JoVE.com

Vi takker Dr. Atsushi Miyawaki og Dr. Richard J. Youle til deling af mt-Keima plasmid og mt-Keima integreret Hela celler. Vi takke Raghavendra A. Shamanna og Dr. Deborah L. Croteau for kritisk læsning af håndskriftet. Denne forskning blev støttet af murene forskningsprogrammet NIH (VAB), tilskud National Institute on aldring, såvel som en 2014-2015 og en 2016-2017 NIA intra-laboratorium (EFF, VAB). EFF blev støttet af HELSE SOR-OST RHF (projekt nr: 2017056) og Norges forskning Rådet (projekt nr: 262175).
FORFATTER BIDRAG:
EFF designet håndskriftet og udarbejdede udkast til; KP, NS, EMF, RDS, JSK, SAC, YH og ED skrev forskellige sektioner af papir; NT, JP, HN og VAB revideret håndskriftet og forudsat ekspertise.

Dyr (mandlige og kvindelige mus) blev født og opdrættet i et akkrediteret Dyrefacilitet i overensstemmelse og godkendelse af NIH Animal Care og brug udvalget. Dødshjælp metoder skal være i overensstemmelse med alle nationale og institutionelle bestemmelser.
1. påvisning af Mitophagy i humane celler
<ol><li>Påvisning af mitophagy ved hjælp af mt-Keima plasmid Bemærk: Keima protein, smeltet til en mitokondrier target signal, forenklet som mt-Keima, har vist sig nyt...

<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. S., Horvath, 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=Mitochondrial+ROS+signaling+in+organismal+homeostasis.">Mitochondrial ROS signaling in organismal homeostasis.</a> Cell. 163 (3), 560-569 (2015).</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=Coordination+of+mitophagy+and+mitochondrial+biogenesis+during+ageing+in+C.+elegans.">Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans.</a> Nature. 521 (7553), 525-528 (2015).</li><li>Lazarou, 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+ubiquitin+kinase+PINK1+recruits+autophagy+receptors+to+induce+mitophagy.">The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.</a> Nature. 524 (7565), 309-314 (2015).</li><li>Kerr, J. S., 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+and+Alzheimer's+Disease:+Cellular+and+Molecular+Mechanisms.">Mitophagy and Alzheimer's Disease: Cellular and Molecular Mechanisms.</a> Trends neurosci. 40 (3), 151-166 (2017).</li><li>Fivenson, E. 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+in+neurodegeneration+and+aging.">Mitophagy in neurodegeneration and aging.</a> Neurochem Int. , (2017).</li><li>Menzies, F. M., Fleming, A., Rubinsztein, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compromised+autophagy+and+neurodegenerative+diseases.">Compromised autophagy and neurodegenerative diseases.</a> Nat Rev Neurosci. 16 (6), 345-357 (2015).</li><li>Rubinsztein, D. C., Shpilka, T., Elazar, Z. <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+autophagosome+biogenesis.">Mechanisms of autophagosome biogenesis.</a> Curr Biol. 22 (1), R29-R34 (2012).</li><li>Kerr, J. S., 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+and+Alzheimer's+disease:+cellular+and+molecular+mechanisms.">Mitophagy and Alzheimer's disease: cellular and molecular mechanisms.</a> Trends neurosci. 40 (3), 151-166 (2017).</li><li>Menzies, F. M., Moreau, K., Puri, C., Renna, M., Rubinsztein, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+autophagic+activity+in+mammalian+cells.">Measurement of autophagic activity in mammalian cells.</a> Curr Protoc Cell Biol. , 16 (2012).</li><li>Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+quantitative+technique+for+detecting+autophagic+events+based+on+lysosomal+delivery.">A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.</a> Chem Biol. 18 (8), 1042-1052 (2011).</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=Measuring+In+Vivo+Mitophagy.">Measuring In Vivo Mitophagy.</a> Mol Cell. 60 (4), 685-696 (2015).</li><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=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>

Nøjagtig måling af mitophagy teknisk krævende. Her har vi præsenteret flere robust teknikker, som giver mulighed for både kvalitativ påvisning af mitophagy og kvantificering af mitophagy niveauer i de mest almindelige laboratorium eksperimentelle modeller.
At erhverve replikerbare data, en eksperimenterende design med mindst tre biologiske gentagelser er nødvendige. Alle forskere er involveret i forsøg og analyser skal være blændet eksperimentelle gruppe identiteter. Derudover skal i...

Mitophagy er afgørende for mitokondrie vedligeholdelse. Mitokondrier gennemskære flere celle signalering veje og er universel subcellulære organeller ansvarlig for produktion af cellulære energi, cellestofskiftet og calcium homeostase1,2,3, 4. mitokondrier konstant opleve udfordringer fra endogene og eksogene kilder, såsom reaktive ilt arter (ROS) og mitokondriel giftstoffer, henholdsvis, der føre til generation af "alderen" og dysfunktionelle mitochondrier. Ophobning af beskadigede mitokondrier formindsker effektiviteten af ATP produktionen samtidig øge mængden af skadelige ROS, og har været forbundet med aldersrelaterede sygdomme som metaboliske sygdomme, annonce, og PD1,5,6 . For at forhindre mitokondrier induceret cellulære dysfunktion, betegnes celler skal specifikt genkender beskadiget mitokondrier og effektivt fjerne dem gennem en cellulær proces mitokondrie autophagy (mitophagy). Det demonstrerede betydningen af mitophagy i sundhed og sygdom illustrerer behovet for præcise og effektive metoder til at opdage mitophagy både in vitro- og in vivo.
Mitophagy er en flertrins proces, der involverer mange proteiner og protein komplekser5,7,8. Kort sagt, er en beskadiget mitochondriet først anerkendt og opslugt af en dobbelt-membraned phagophore, som kan stamme fra plasma membran, endoplasmatiske reticulum, Golgi kompleks, kernen eller mitokondriet selv9,10. Den sfæriske phagophore elongates og til sidst sæler mitokondrier inde, der udgør den mitokondrielle autophagosome (mitophagosome). Mitophagosome derefter sikringer med lysosomet for nedbrydning, danner en autolysosome, hvori den beskadigede mitochondriet er nedbrudt og genanvendt7,8. Store autophagic proteiner også involveret i mitophagy omfatter: Autophagy relaterede 7 (ATG7) og Beclin1 (indledning), Microtubule-Associated Protein 1A/1B-lys kæde 3 (LC3-II) (LGG-1 i C. elegans) og p62 (komponenter i phagophore), og lysosomale-associerede membran glycoprotein 2 (LAMP2)6,7. Derudover er der flere væsentlige proteiner unikke til mitophagy, herunder PT-induceret formodede Kinase 1 (PINK-1), Parkin1, nukleare Dot Protein 52 kDa (NDP52), optineurin, BCL2 interagere Protein 3 som (NIX/BNIP3L) (DCT-1 i C. elegans), blandt andre5,6,11.
En fælles metode til at registrere ændringer i niveauet af autophagy er af forholdet mellem LC3-II/LC3-jeg eller LC3-II/aktin. Men denne metode er uspecifikke, som en forøgelse af dette forhold kan afspejle enten en øget indledning eller en nedsat fusion af mitophagosome at lysosomet12. En anden metode er at vurdere colocalization mellem en autophagy markør (fxLC3) og en mitokondrie protein (f.eks.Translocase af ydre mitokondrielle membran 20 (TOMM20, som kunne blive forringet af proteasomes)). Men dette kan kun angive ændringer i samlede mitophagy niveauer og ikke kan skelne trin på hvilke blokering opstår. Dette kan afklares ved hjælp af lysosomale hæmmere (f.eks.E64d + Pepstatin A, kaldt EP) i parallel til at forårsage en ophobning af mitophagosomes. Forskellen mellem antallet af mitophagosomes ved baseline og antallet af mitophagosomes efter behandling med EP kan angive mitophagy. Disse begrænsninger har bedt om udviklingen af roman mitophagy detektionsteknikker. I betragtning af den stigende betydning af mitophagy i et bredt spektrum af sygdomme hos mennesker præsenterer vi flere robuste mitophagy detektionsteknikker, som kan være nyttige for forskere. Vi dækker både in vitro- og i vivo teknikker og anbefaler at kombinere flere teknikker for at kontrollere ændringer af 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

Mitokondrier er kraftcentre af celler og producerer cellulære energi i form af ATP. Mitokondriel dysfunktion bidrager til biologiske aldring og en bred vifte af lidelser, herunder metaboliske sygdomme, for tidlig aldring syndromer og neurodegenerative sygdomme såsom Alzheimers sygdom (AD) og Parkinsons sygdom (PD). Vedligeholdelse af mitokondrie sundhed afhænger af mitokondriel Biogenese og effektiv regnskabsafslutning af dysfunktionelle mitochondrier gennem mitophagy. Eksperimentelle metoder til præcist afsløre autophagy/mitophagy, især i dyremodeller, har udfordrende for at udvikle. De seneste fremskridt i retning af forståelse for de molekylære mekanismer af mitophagy har muliggjort udviklingen af roman mitophagy detektionsteknikker. Her, vi introducere flere alsidig teknikker til at overvåge mitophagy i humane celler, Caenorhabditis elegans (fxRosella og DCT-1 / LGG-1 stammer), og mus (mt-Keima). En kombination af disse mitophagy detektionsteknikker, herunder cross-arter evaluering, vil forbedre nøjagtigheden af mitophagy målinger og føre til en bedre forståelse af rollen, som mitophagy i sundhed og sygdom.

Påvisning af Mitophagy i humane celler:
Ved hjælp af den procedure, der præsenteres her, blev menneskelige HeLa celler transfekteret med mt-Keima plasmid. Raske celler viste et velorganiseret mitokondrie netværk (normal god landbrugspraksis, 488 nm) med få forekomster af mitophagy (RFP, 561 nm). Dog celler forbehandlet med en mitokondrie uncoupler FCCP (30 µM til 3 h) udstillede en dyb stigning i mitophagy forekomst (<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

In Vitro og In Vivo påvisning af Mitophagy i humane celler, C. Elegansog mus

Mitophagy, processen med clearing beskadiget mitokondrier, er nødvendige for mitokondrie homøostase og sundhed vedligeholdelse. Denne artikel præsenterer nogle af de nyeste mitophagy detektionsmetoder i humane celler, Caenorhabditis elegansog mus.

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

Research

JoVE Journal

Medicine

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

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stamcelletransplantation i en In vitro Simuleret Iskæmi / reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

Halspulsåren infusioner farmakokinetisk og farmakodynamisk analyse af Taxaner i mus

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Evaluere effektiviteten af ​​Cancer Drug Sensibilisering<em&gt; In vitro</em&gt; Og<em&gt; In Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Intrauterin Telemetri måler Mouse kontraktile Pressure<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Studere i bugspytkirtlen Kræft stamceller Karakteristik for udvikling af nye behandlingsstrategier

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Generation af Microtumors Brug 3D Menneskelig Biogel Kultur System og Patient-afledte glioblastomaceller for Kinomic Profilering and Drug respons Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

Udtømning af Mouse Celler fra Humane tumorxenoplantater Markant Forbedrer Downstream Analyse af Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Optimeret Analyse af<em&gt; In vivo</em&gt; og<em&gt; In vitro</em&gt; Nedsat Steatose

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

In Vivo Photolabeling af celler i tyktarmen til at vurdere vandrende potentiale af hæmatopoietisk celler i Neonatal mus

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...