Eine Vielzahl von Luciferase exprimierenden Zelllinien können für die präklinische Forschung in Maus-Modellen verwendet werden. Diese Zellen werden als pathogen-freien gefrorenen Kultur, die sich leicht in Standard-Medien wachsen, ohne dass für Selektionsmarker zur Verfügung gestellt. Für unser Experiment verwenden wir die 4T1-luc2 murine Brusttumor-Zelllinie, die das Luciferase-Gen, das als eine optische Anzeige der Genexpression oder Tumorgenese in vivo dient zum Ausdruck bringt. Wir verwenden Luciferaseexpression zum Wachstum des Primärtumors nicht-invasiv Spur, aber sie können auch verwendet werden, zu lokalisieren und zu überwachen metastatischen Läsionen werden. Luciferaseaktivität überprüft werden müssen vor der Injektion, und um dies tun zu können, ist eine 90% konfluent Kolben durch Trypsinierung geerntet und dann gezählt. 50.000 Zellen werden dann in einer einzigen Vertiefung einer Mikrotiterplatte und Verdünnungsreihen verzichtet werden durchgeführt. Luciferin wird in die Vertiefungen an 150ug pro ml zugegeben und für 2 Minuten. Die Mikrotiterplatte kann in IVIS oder einer lumineszierenden Platte Leser Expression bestimmen abgebildet werden. Diese Zellinie exprimiert bis 6500 Photonen pro Sekunde pro Zelle, aber jeder Ausdruck Ebene oberhalb von 500 Photonen pro Sekunde pro Zelle können erfolgreich in vivo dargestellt werden. Jetzt, da wir Zellen optimale Aktivität haben, können wir auf die subkutane Injektion Schritt fort. Um eine optimale Erkennung des Tumors zu erleichtern, werden wir die Verwendung eines athymischen immungeschwächten Nacktmaus Belastung. Vor der Injektion werden die Tiere betäubt, um für tiefe Betäubung Wirkung. Als nächstes werden wir spritzen bis zu 250.000 Zellen in 100 ul PBS subkutan in die Flanke injiziert. Laden Sie die Zellen in einer 1 ml Spritze eine 26 Gauge Nadel. Heben Sie die Haut vorsichtig mit einer Pinzette ein &quot;Zelt&quot; zu machen und injizieren die Zellen an der Basis. Die neu injizierten Zellen können sofort abgebildet werden. In jedem Imaging-Sitzung insgesamt 150 mg Luciferin pro kg Körpergewicht wird dann über zwei Injektionen in die Bauchhöhle verabreicht. In dieser Studie werden die Tiere 10 Minuten nach Injektion Luciferin abgebildet, um konsistente Photonenfluss zu gewährleisten. Wir zeigen Ihnen, diese im nächsten Schritt. Luciferin Kinetik von Tag zu Tag variieren. In diesem Modell, Messung 10 Minuten nach Injektion Luciferin gab ein Ergebnis innerhalb eines Bereiches von 15% Variabilität. Für unser Experiment werden wir die IVIS Spectrum in vivo Imaging-System verwenden wird ein Back-ausgedünnt charge coupled device gekühlt auf -90 ° C bis maximale Empfindlichkeit zu erreichen. Zur Unterstützung der absolute Quantifizierung, misst das System dunklen Ladung während down-Zeit und führt eine Selbstkalibrierung während der Initialisierung. So starten Bildgebung, initialisieren Sie die IVIS System (ein Klick) und stellen Sie die Imaging-Parameter für das Experiment. Wählen Sichtfeld für die Anzahl der Tiere abgebildet. Bis zu 5 Tiere können in das Gerät mit dem integrierten Anästhesie vielfältig gepflegt werden. Die Bühne ist mit einer konstanten 37 ° C auf Körpertemperatur der Tiere aufrecht zu erhalten. Ein EKG-Anschluss ist vorgesehen, um die Tiergesundheit in stressigen Verfahren zu überwachen. In Living Image Software, Belichtungszeit, Blende und Pixel-Binning können basierend auf der Expression der Zelllinie optimiert werden. Diese Einstellungen können jederzeit während eines Experiments werden ohne Auswirkungen auf das quantitative Ergebnis verändert. IVIS erwirbt ein fotografisches Bild des Tieres unter weißem Licht und einer quantitativen Biolumineszenz oder Fluoreszenz-Signal, das auf dem Bild überlagert ist. Die Biolumineszenz-Signal wird in Photonen pro Sekunde und als einer Intensität Karte. Die Bildanzeige wird angepasst, um optimale Kontrast und Auflösung des Bildes, ohne die Quantifizierung bieten. Lumineszenz aus den Zellen können an der Injektionsstelle mit einem region of interest-Tool gemessen werden. Die Messdaten werden in der Tabelle zusammen mit allen experimentellen Parameter im Zusammenhang mit der Aufnahme von Bildern, die gespeichert oder exportiert werden kann für die Analyse angezeigt Mehrere Bilder können erworben werden und im Vergleich in Längsschnittstudien für Sekunden oder Monaten, je nach der Art des Experiments. Wir werden Photonenfluss aus dem Tumor zu messen zum Zeitpunkt Null und Monitor für 4 Wochen mit Bildgebung bei bi-wöchentlichen Abständen. Photonenfluss aus dem Tumor ist proportional zur Anzahl der lebenden Zellen, die Luciferase so Biolumineszenz korreliert direkt mit der Tumorgröße. Bei 5 Tage nach der Implantation des Tumors ist noch nicht greifbar, aber die Zellen können durch Biolumineszenz und der Tumor gesehen werden aktiv wachsenden quantifiziert werden. In diesem Stadium der Biolumineszenz-Signal ist viel stronger. Die Belichtungszeit, Blende und Pixel-Binning kann so eingestellt, dass das Bild klar ist und die Kamera nicht zu sättigen. IVIS kompensiert automatisch die Änderungen im Lichte Sammlung, so dass diese Messungen mit denen früher und später im Experiment gesammelten verglichen werden kann. An 7 Tagen nach der Implantation Tumoren sind spürbar zum ersten Mal und Biolumineszenz-Messung bereits 7 Tage der Daten erzeugt hat. Nach 28 Tagen nach der Implantation, sind Tumoren es nekrotisch und Zellen beginnen zu sterben. Tumorgröße durch Dickenmessung geschätzt nicht merklich verändert, aber Lumineszenz aus dem Tumor verringert Angabe Zelltod. Caliper Messungen und Biolumineszenz-Messung kann fortgesetzt werden, bis eine humane Endpunkt erreicht ist. Tumor-Nekrose durch Hypoxie oder Behandlungen wird durch reduzierte Biolumineszenz angegeben werden, auch wenn sie reduzieren nicht die Tumormasse.

Ich möchte versichern, dass alle Experimente durchgeführt, um die Zeitschrift Visualisierte Experimente Artikel produzieren im Einklang waren mit dem Tier-Protokoll 060 vom Institutional Animal Care genehmigt durchgeführt und Verwenden Committee (IACUC) bei Caliper Life Sciences, Alameda CA Jae Kim, Ph . D., IACUC Chair, Caliper Life Sciences.

in vivo bioluminescent imaging of mammary tumors using ivis spectrum

4T1 Maus Mamma Tumorzellen subkutan in nu / nu-Mäusen implantiert werden, um tastbare Tumoren in 15 bis 20 Tage bilden. Diese Xenograft-Tumor-Modell ist für die präklinische in vivo Evaluierung von mutmaßlichen Antitumorverbindungen wertvoll. Die 4T1-Zelllinie wurde entwickelt, um konstitutiv die Glühwürmchen-Luciferase-Gen (luc2). Wenn Mäuse mit 4T1-luc2 Tumoren sind mit Luciferin die Tumoren strahlen eine visuelle Lichtsignal, überwacht mit Hilfe eines empfindlichen optischen Imaging-System wie die IVIS Spectrum kann injiziert. Der Photonenfluss aus dem Tumor ist proportional zur Anzahl der lichtemittierenden Zellen und das Signal kann gemessen werden, um das Tumorwachstum und die Entwicklung zu überwachen. IVIS kalibriert ist, damit absolute Quantifizierung der Biolumineszenz-Signal und Längsschnittstudien über viele Monate und über mehrere Aufträge von Signalgröße kann, ohne dabei das quantitative Ergebnis durchgeführt werden. Das Tumorwachstum kann für mehrere Tage durch Biolumineszenz beobachtet werden, bevor die Tumorgröße wird spürbar oder messbar traditionellen physikalischen Mitteln. Diese schnelle Überwachung kann Einblick in frühe Ereignisse in der Tumor-Entwicklung bereitzustellen oder führen zu kürzeren experimentellen Verfahren. Tumor Zelltod und Nekrose durch Hypoxie oder medikamentöse Behandlung ist eine frühzeitige durch eine Reduktion der Biolumineszenz-Signal angezeigt. Das Zelltod möglicherweise nicht durch eine Reduktion der Tumorgröße von materiellen Mitteln gemessen begleitet werden. Die Fähigkeit zur frühen Ereignisse in Tumor-Nekrose-sehen hat erhebliche Auswirkungen auf die Auswahl und Entwicklung von Therapeutika. Quantitative Bildgebung des Tumorwachstums mit IVIS bietet eine präzise Quantifizierung und beschleunigt den experimentellen Prozess, um Ergebnisse zu erzielen.

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

Brusttumor-Zellen, die Luciferase werden subkutan in Mäuse implantiert und visualisiert mit Hilfe der optischen Bildgebung, das Tumorwachstum und die Entwicklung nicht-invasiv in einer Längsschnittstudie zu überwachen.

In vivo Biolumineszenz Imaging von Brusttumoren mit IVIS Spectrum

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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In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

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Serie: 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 Photoschalten durch die Mammary Imaging Fenster

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

Forschung

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.

In-vivo-Imaging von Deep kortikalen Schichten mit einem MICROPRISMEN

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.

Mesoscopic Fluoreszenz-Tomographie für In-vivo Imaging der Entwicklungsländer Drosophila</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.

Brustepithelzellen Transplantation

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.

Imaging Protein-Protein-Wechselwirkungen 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.

Einzel-Molekül-Imaging der Genregulation<em&gt; In vivo</em&gt; Verwenden cotranslationalen Aktivierung durch Spaltung (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-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

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.

gDNA Enrichment durch eine Transposase-basierte Technologie für NGS-Analyse der gesamten Sequenz von<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt; Und 9 Gene in der DNA-Reparatur beteiligt Schäden

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.

Ein screenbaren<em&gt; In-vivo-</em&gt; Assay für Mitochondriale Modulatoren Verwendung transgener 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.

Messung der Steifigkeit von<em&gt; Ex Vivo</em&gt; Maus Aorten Mit Rasterkraftmikroskopie

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