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.

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The Bohr laboratory has CRADA arrangements with ChromaDex and GlaxoSmithKline.

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 must be randomly chosen during image acquisition. For cell culture and C. elegans studies, at least three biological repeats should be performed. For mt-Keima mouse studies, it is recommended to use a sufficient number of mice to achieve statistical significance. For mammalian cells, analyze 30-100 cells for each experiment, and run at least 3 different experiments. Quality control at these steps enables replicable results. Detection of mitophagy in yeast has recently been summarized elsewhere25.
There are several critical steps in the in vitro mitophagy detection techniques. Transfection efficiency, which depends on the quality of reagents and the DNA used, is vital during transfection of the mt-Keima protein. Thus, optimization of the transfection efficiency in different conditions (e.g., using different cell lines) is necessary. For the LAMP2/COXII method, antibody specificity and optimal primary antibody dilution fold affect the quality of the images and should be tested before beginning experiments. The same applies to the high-content imaging method, where antibody quality and dilution are crucial. The automated microscopy system and the customized analysis protocol allows for the imaging and analysis of at least 1,500 cells/condition, which makes the results extremely robust compared to other in vivo imaging based methods. Additionally, the analysis protocol provides data on further mitochondrial parameters such as length and total area on a cell by cell basis, which is highly useful in mitophagy research.
In some cases, it is not recommended to assay the colocalization of LAMP2 with mitochondrial outer membrane proteins such as TOMM20. The very early stages of mitophagy involve Parkin-mediated ubiquitination of mitochondrial outer membrane proteins, including TOMM20 and Mitofusin 1 (MFN1). Parkin conjugates multiple different ubiquitin chain linkages on these proteins including K48-linked ubiquitin chains, which promote degradation of the protein by the 26S proteasome. Therefore, these ubiquitinated mitochondrial outer membrane proteins can still be degraded even though mitophagy is blocked6. This can skew the data, resulting in false positives in overall data interpretation. In addition, elimination of mitochondrial DNA (mtDNA nucleoids) is a second indicator of mitophagy, and it can be quantified by immunofluorescence using an anti-DNA antibody6.
Some critical steps for monitoring in vivo mitophagy in C. elegans are listed below:
1. Transfer of transgenic animals to new plates every 24&#8211;48 h is recommended to avoid the outgrowth of progeny, which leads to a mixed population or starvation, which itself may trigger mitophagy. Therefore, non-starved nematodes should be used.
2. Levamisole is a mild anesthetic which is used to immobilize nematodes. Avoid anesthetics that could affect mitochondrial activity, such as sodium azide. Sodium azide blocks components of the mitochondrial respiratory chain and perturbs energy generation, leading to mitochondrial and oxidative stress. Thus, sodium azide is likely to trigger mitophagy.
3. Specimens should not be allowed to dry out during microscopic examination. Therefore, the use of M9 buffer instead of water is recommended to ensure favorable osmotic conditions.
4. Intestinal autofluorescence increases with age in C. elegans. Thus, body-wall muscle cells close to the intestine should be avoided during microscopic examination.
5. If paraquat fails to induce mitophagy: a. increase paraquat exposure time on worms, b. increase paraquat concentration, or c. prepare a fresh working solution of paraquat since its efficiency declines over time.
6. If increased matricidal hatching is observed in worms exposed to paraquat, either: a. reduce the period of paraquat treatment, b. reduce paraquat concentration, c. supplement plates with fluorodeoxyuridine (FUdR) (final concentration of 100-400 &#956;M), an inhibitor of DNA synthesis that prevents egg hatching, or d. conduct the experiment in older worms (e.g., 4-day-old worms).
This procedure describes the steps for imaging mitophagy in the liver using the mt-Keima transgenic mice, as shown in Figure 3. Tissues other than the brain and liver have been analyzed using the mt-Keima system14. Dual-excitation ratio imaging in liver tissues is obtained via two sequential excitations. All images must be obtained from freshly excised organs. Tissues examined should be kept cold and processed as quickly as possible. Liver slices can be obtained at any age. When imaging mitophagy, optimize laser powers and exposure times for each organ during microscopic analysis. Laser power should be set at the lowest output that allows clear visualization of the mt-Keima signal14. However, there are some limitations for the mt-Keima transgenic mice. Due to the instability of Keima as well as the loss of a pH gradient across the lysosomal membrane under fixation, this mouse model is not suitable for cryo-sectioning and immunohistochemistry. Another limitation is that mt-Keima, or matrix aggregates of this protein, may affect unknown mitochondrial or cellular functions, even though it does not change mitochondrial oxygen consumption rate14. Furthermore, the time and cost associated with mt-Keima mice render it less desirable for high-throughput analysis than the aforementioned cell culture and C. elegans methods. Besides the methods mentioned here, other ways to quantitatively study mitophagy in mt-Keima mice include Western blot or FACS. To note, in addition to the mt-Keima transgenic mice, there is another mitophagy mouse model, the &#34;mito-QC&#34;, a transgenic mouse model with a pH-sensitive fluorescent mitochondrial signal26. Due to the complexity and dynamics of mitophagy, we recommend combining the aforementioned fluorescence methods with other common mitophagy detection methods, such as electron microscopy and Western blotting, to strengthen the findings.
The development of new mitophagy detection techniques such as those mentioned here will have broad applications, from mechanistic studies of mitophagy to high throughput drug screens. It should be noted that mitophagy is easily affected by both endogenous and exogenous fluctuations (e.g., cell culture conditions such as cell density, starvation, hypoxia, etc.)1,5,8. Therefore, it is paramount that the same experimental conditions are maintained for all experiments. It is important to use a combination of different mitophagy detection methods in both in vitro and in vivo studies to verify the correct interpretation of mitophagy status. Further understanding of the mechanisms of mitophagy, achieved through techniques mentioned herein, will allow for the development of new, more precise methods of mitophagy detection.

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&lt;br/&gt; 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&amp;ndash;Dulbecco&#039;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[p&lt;em&gt;myo-3&lt;/em&gt; TOMM-20::Rosella]</td><td>Material inquiry to Tavernarakis Nektarios</td><td></td><td>Maintain transgenic animals at 20 &amp;deg;C</td></tr><tr><td>IR: N2; Ex[p&lt;em&gt;dct-1&lt;/em&gt; DCT-1::GFP; p&lt;em&gt;myo-3&lt;/em&gt; DsRed::LGG-1]</td><td>Material inquiry to Tavernarakis Nektarios</td><td></td><td></td></tr><tr><td>p&lt;em&gt;myo-3&lt;/em&gt; TOMM-20::Rosella</td><td>Material inquiry to Tavernarakis Nektarios</td><td></td><td></td></tr><tr><td>p&lt;em&gt;dct-1&lt;/em&gt; DCT-1::GFP</td><td>Material inquiry to Tavernarakis Nektarios</td><td></td><td></td></tr><tr><td>p&lt;em&gt;myo-3&lt;/em&gt; 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 &amp;deg;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 &amp;ndash; 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). Mitophagy was also measured using the co-localization between LAMP2 and COXII (Figure 1B).
Detection of Mitophagy in C. Elegans
We examined the body-wall muscle cells of transgenic nematodes expressing DsRed fused with LGG-1 and either mtRosella or DCT-1 fused with GFP under normal and mitophagy-inducing conditions such as oxidative stress. Transgenic animals were exposed to paraquat (8 mM for 2 days). Mitophagy induction is indicated by the decreased GFP/DsRed ratio of mtRosella fluorescence (Figure 2A). Additionally, the elevated number of co-localization events between DCT-1::GFP and DsRed::LGG-1 signals signifies mitophagy stimulation in response to oxidative stress (Figure 2B).
Detection of Mitophagy Using the mt-Keima Mice:
Imaging analysis of in vivo mitophagy provides insight into mitophagy in normal and pathophysiological conditions. Using the protocol described above, mitophagy occurrence in different organ tissues, such as liver and cerebellum (Figure 3), can be visualized with confocal mitophagy.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56301/56301fig1.jpg" /> 
Figure 1. Different methods to evaluate mitophagy in human cells. (A) Human HeLa cells were transfected with mt-Keima plasmid for three days, followed by imaging at both 488 nm and 561 nm. As a positive control, ceflls were treated with FCCP (30 &#181;M) 3 h prior to imaging. (B) Images of human primary fibroblasts stained with LAMP2 (RFP) and COXII (GFP). (C) Images of human fibroblasts stained with anti-TOM20 (red) and anti-LC3 (green) antibodies. The bars on the right show the readout of mitochondria co-localizing with autophagosomes. Co-localization is increased by energetic stress (glucose-free galactose media) and the addition of lysosomal inhibitors E64d and Pepstatin A (EP) for 24 h. Representative scale bars for (A), (B), and (C) are labeled on the last figure of each panel, independently. Quantitative data are shown in mean &#177; S.E.M. ***p &#60; 0.001; t-test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56301/56301fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56301/56301fig2.jpg" /> 
Figure 2. In vivo detection of mitophagy in C. elegans. (A) Transgenic nematodes expressing mtRosella in body-wall muscle cells were treated with 8 mM paraquat. Mitophagy stimulation is signified by the decreased ratio between pH-sensitive GFP to pH-insensitive DsRed (n = 50; ***p &#60; 0.001; t-test). Arrowheads point out intestinal autofluorescence. Scale bars denote 20 &#181;m. Acquisition details: Exposure time, 200 ms; Contrast, medium. Images were acquired using a 10X objective lens. Quantitative data are shown in mean &#177; S.E.M. values. (B) Transgenic nematodes co-expressing mitophagy receptor DCT-1 fused with GFP together with the autophagosomal protein LGG-1 fused with DsRed in body-wall muscle cells were exposed to 8 mM paraquat. Mitophagy induction is signified by co-localization of GFP and DsRed signals (for each group of images DCT-1 is shown in green on top, autophagosomes in red below, and a merged image at the bottom; n = 30; ***p &#60; 0.001; t-test). Sale bars denote 20 &#181;m. Acquisition details: Resolution, 1,024 X 1,024; Master gain, Track1: 562 and Track2: 730; Emission filters, Track1 Channel1: 639 nm and Track2 Channel2: 519 nm; Laser intensity, Track1 (543 nm): 12.9% and Track2 (488 nm): 39.4%. Images were acquired using a 40X objective lens. Quantitative data are shown in mean &#177; S.E.M. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56301/56301fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56301/56301fig3.jpg" /> 
Figure 3. In vivo detection of mitophagy in mt-Keima transgenic mice. Representative images of mt-Keima signals in the liver (upper) and cerebellum area (including Purkinje cells, lower panel) of a mt-Keima mouse (458 nm, green; 561 nm, red). Scale bar denotes 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56301/56301fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

17.5K Views.  National Institutes of Health. Mitophagy, het proces van clearing beschadigd de mitochondriën, is noodzakelijk voor mitochondriale homeostase en gezondheid onderhoud. Dit artikel presenteert een aantal van de meest recente mitophagy detectiemethoden in menselijke cellen, Caenorhabditis elegansen muizen.

Video: 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 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 ...

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 ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Biology

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

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

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

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

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. ...

Quantifying Mitophagy in C. elegans and a Liver Cancer Cell Line

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Author Spotlight: Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

Quantitative Analysis of Mitophagy in Murine Pancreatic &#946;-Cells

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Author Spotlight: Assessment of Mitophagy Flux in Pancreatic &#946;-Cells Using Effective and Robust Complementary Approaches 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes

Source: Fang, E. F., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56301">In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice.</a> J. Vis. Exp. (2017).
This video demonstrates an in vivo assay to study mitophagy in Caenorhabditis elegans. Upon exposing the nematodes to a mitophagy-inducing drug, the occurrence of mitophagy is assessed by co-localization between DCT-1, a mitophagy receptor on the damaged mitochondria, and the autophagosomal membrane protein LGG-1.

1. Detection of Mitophagy in C. elegans
NOTE: The nematode C. elegans provides a platform to assay mitophagy at the organismal level. Two strains can be ...

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

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