Une grande variété de luciférase exprimant des lignées cellulaires cancéreuses peuvent être utilisés pour la recherche pré-clinique dans des modèles murins. Ces cellules sont fournies en tant que culture exempts d&#39;agents pathogènes congelés, qui volontiers se développer dans les médias standards sans besoin de marqueurs de sélection. Pour notre expérimentation, nous allons utiliser le 4T1-luc2 lignée murine de cellules tumorales mammaires, qui exprime le gène de la luciférase qui sert comme un indicateur optique de l&#39;expression des gènes ou de tumorigenèse in vivo. Nous allons utiliser l&#39;expression de luciférase pour suivre la croissance de la tumeur primaire non invasive, mais ils peuvent aussi être utilisées pour localiser et surveiller les lésions métastatiques. L&#39;activité luciférase doit être vérifiée avant l&#39;injection, et pour ce faire, une fiole de 90% de confluence est récoltée par trypsinisation puis comptés. 50.000 cellules sont ensuite distribués dans un seul puits d&#39;une plaque de microtitration et dilutions sont effectuées. Luciférine est ajoutée aux puits à 150ug par ml et incubé pendant 2 minutes. La microplaque peuvent être visualisées dans IVIS ou un lecteur de plaque luminescente afin de déterminer les niveaux d&#39;expression. Cette lignée cellulaire exprime jusqu&#39;à 6500 photons par seconde par cellule, mais aucun niveau d&#39;expression de plus de 500 photons par seconde par cellule peut être imagée avec succès in vivo. Maintenant que nous avons des cellules de l&#39;activité optimale, nous pouvons passer à l&#39;étape d&#39;injection sous-cutanée. Afin de faciliter la détection optimale de la tumeur, nous utilisons une souche athymiques immunodéprimés souris nude. Avant l&#39;injection, les animaux sont anesthésiés à l&#39;effet d&#39;une anesthésie profonde. Ensuite, nous allons injecter jusqu&#39;à 250 000 cellules dans 100 ul de PBS sont injectés par voie sous cutanée dans le flanc. Chargez les cellules dans une seringue de 1 ml et fixer une aiguille de calibre 26. Soulevez délicatement la peau avec une pince pour faire une &quot;tente&quot; et d&#39;injecter les cellules à la base. Les cellules nouvellement injectés peuvent être visualisés immédiatement. À chaque séance d&#39;imagerie un total de 150mg de luciférine par kg de poids corporel est alors administrée par deux injections dans la cavité péritonéale. Dans cette étude, les animaux sont imagés 10 minutes après l&#39;injection de luciférine pour assurer le flux de photons cohérents. Nous allons vous montrer ce à l&#39;étape suivante. Cinétiques luciférine peut varier de jour en jour. Dans ce modèle particulier, mesurant 10 minutes après l&#39;injection luciférine a donné un résultat dans une fourchette de variabilité de 15%. Pour notre expérience, nous allons utiliser le spectre IVIS l&#39;imagerie in vivo système utilise un arrière-aminci charge coupled device refroidi à -90 ° C pour obtenir une sensibilité maximale. Pour soutenir la quantification absolue, le système des mesures de charge obscurité pendant les temps d&#39;arrêt et exécute une auto-étalonnage lors de l&#39;initialisation. Pour commencer l&#39;imagerie, initialiser le système IVIS (un clic) et définissez les paramètres d&#39;imagerie pour l&#39;expérience. Sélectionnez le champ de vision pour le nombre d&#39;animaux étant imagé. Jusqu&#39;à 5 animaux peuvent être maintenus dans l&#39;instrument en utilisant le collecteur intégrante anesthésique. La scène est à 37 ° C à constamment maintenir la température corporelle chez les animaux. Un port ECG est fourni pour surveiller la santé des animaux pendant les procédures stressant. Dans Vivre logiciel Image, temps d&#39;exposition, f-stop et pixel binning peut être optimisée en fonction du niveau d&#39;expression de la lignée cellulaire. Ces paramètres peuvent être modifiés à tout moment lors d&#39;une expérience sans impact sur le résultat quantitatif. IVIS acquiert une image photographique de l&#39;animal sous une lumière blanche et un signal quantitatif bioluminescent ou fluorescent, qui est superposée sur l&#39;image. Le signal de bioluminescence est exprimé en photons par seconde et affichée comme une carte d&#39;intensité. L&#39;affichage de l&#39;image est ajustée pour fournir un contraste optimal et la résolution de l&#39;image sans affecter la quantification. Luminescence des cellules peut être mesurée à l&#39;endroit de l&#39;injection en utilisant une région de l&#39;outil d&#39;intérêt. Les données de mesure sont affichées dans le tableau avec tous les paramètres expérimentaux relatifs à la capture d&#39;images qui peuvent être enregistrés ou exportés pour une analyse Plusieurs images peuvent être acquises et comparées dans les études longitudinales couvrant secondes ou quelques mois selon la nature de l&#39;expérience. Nous allons mesurer le flux de photons provenant de la tumeur au temps zéro et moniteur pour 4 semaines avec l&#39;imagerie au bi-hebdomadaire d&#39;intervalle. Flux de photons provenant de la tumeur est proportionnelle au nombre de cellules vivantes exprimant la luciférase pour la bioluminescence est directement corrélée avec la taille des tumeurs. A 5 jours après l&#39;implantation de la tumeur n&#39;est pas encore palpables, mais les cellules peuvent être quantifiés par bioluminescence et la tumeur est considérée être en croissance active. A ce stade, le signal de bioluminescence est beaucoup StrongeR. Le temps d&#39;exposition, f-stop et de pixel binning peut être ajustée de sorte que l&#39;image est claire et la caméra ne sature pas. IVIS compense automatiquement les changements dans la collecte de lumière de sorte que ces mesures peuvent être comparées à celles recueillies précédemment et, plus tard dans l&#39;expérience. À 7 jours après l&#39;implantation des tumeurs sont palpables pour la première fois et la bioluminescence de mesure a déjà généré de 7 jours de données. A 28 jours après l&#39;implantation, les tumeurs deviennent nécrotiques et les cellules commencent à mourir. La taille des tumeurs estimée par la mesure étrier ne change pas sensiblement, mais la luminescence de la tumeur diminue indiquant la mort cellulaire. Mesures étrier et la mesure de la bioluminescence peut être poursuivi jusqu&#39;à ce qu&#39;un critère d&#39;évaluation humanitaire est atteint. Nécrose tumorale due à l&#39;hypoxie des régimes ou des traitements sera indiqué par bioluminescence réduite, même si elles ne réduisent pas la masse tumorale.

Je tiens à assurer que toutes les expériences effectuées pour produire le Journal de l&#39;article Expériences visualisé ont été effectuées en conformité avec le Protocole de 060 animaux approuvé par le soin des animaux et des institutionnels Utilisez Comité (IACUC) à Caliper Life Sciences, Alameda CA Jae Kim, Ph . D., président IACUC, Caliper Life Sciences.

in vivo bioluminescent imaging of mammary tumors using ivis spectrum

4T1 cellules tumeur mammaire de souris peut être implantée sous-cutanée dans la souris nu / nu pour former des tumeurs palpables dans 15 à 20 jours. Ce système de tumeur xénogreffe modèle est précieux pour le pré-cliniques dans l&#39;évaluation in vivo de composés antitumoraux putatifs. La lignée cellulaire 4T1 a été conçu pour exprimer le gène constitutivement la luciférase de luciole (luc2). Lorsque des souris porteuses de tumeurs 4T1-luc2 sont injectés avec luciférine les tumeurs émettent un signal de lumière visible qui peut être surveillé à l&#39;aide d&#39;un système d&#39;imagerie optique sensible comme le spectre IVIS. Le flux de photons provenant de la tumeur est proportionnelle au nombre de cellules émettrices de lumière et le signal peut être mesuré pour contrôler la croissance tumorale et le développement. IVIS est calibré pour permettre la quantification absolue du signal de bioluminescence et les études longitudinales peuvent être réalisées sur plusieurs mois et sur plusieurs ordres de grandeur du signal sans compromettre le résultat quantitatif. La croissance tumorale peut être surveillé pendant plusieurs jours par bioluminescence avant la taille de la tumeur devient palpable ou mesurables par des moyens physiques traditionnels. Ce contrôle rapide peut donner un aperçu des événements précoces dans le développement tumoral ou conduire à des procédures plus courtes expérimental. Mort des cellules tumorales et la nécrose due à l&#39;hypoxie ou le traitement médicament est indiqué au début par une réduction dans le signal de bioluminescence. Cette mort cellulaire pourrait ne pas être accompagnée d&#39;une réduction de la taille tumorale telle que mesurée par des moyens physiques. La capacité de voir les événements précoces de nécrose tumorale a un impact significatif sur la sélection et le développement d&#39;agents thérapeutiques. Imagerie quantitative de la croissance tumorale en utilisant IVIS fournit quantification précise et accélère le processus expérimental pour produire des résultats.

Watch this Scientific Journal Video about In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum at JoVE.com

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Cellules mammaires tumorales exprimant la luciférase sont implantés sous la peau chez la souris et visualisées à l&#39;aide d&#39;imagerie optique pour surveiller la croissance des tumeurs et le développement de façon non invasive dans une étude longitudinale.

In vivo Bioluminescent imagerie des tumeurs mammaires qui utilisent des fréquences IVIS

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

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Research

JoVE Journal

Biology

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

46.9K vues.  Caliper Life Sciences.  中文 

Vidéo: In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

Dendra2 photocommutation travers la fenêtre d&#39;imagerie mammaire

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

Recherche

Biologie

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

L&#39;imagerie in vivo de Deep couches corticales utilisant un microprisme

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mésoscopique fluorescence tomographie pour In vivo L&#39;imagerie du développement Drosophile</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

Mammaires épithéliales procédure de greffe

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imagerie interactions protéine-protéine In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Une seule molécule d&#39;imagerie du règlement Gene<em&gt; In vivo</em&gt; Utilisation de l&#39;activation par clivage cotraductionnelle (COTRAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

ADNg enrichissement par une technologie basée Transposase-NGS Analyse de la séquence plénier de<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt;, et 9 gènes impliqués dans la réparation des dommages de l&#39;ADN

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

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.

Un criblables<em&gt; In Vivo</em&gt; Essai de modulateurs mitochondriale utilisant transgénique Bioluminescent<em&gt; Caenorhabditis elegans</em

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

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Mesure de la rigidité de<em&gt; Ex Vivo</em&gt; Souris aortes Utiliser Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...