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

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>
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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.

in-vitro-vivo-detection-mitophagy-human-cells-c-elegans

Research

JoVE Journal

Medicine

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 time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

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 protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

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

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

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

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 model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

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

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

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

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

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

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 patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

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

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

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

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

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

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

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

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...