Lusiferaz ifade kanser hücre hatları çok çeşitli fare modelleri, ön-klinik araştırma için kullanılabilir. Bu hücreler kolayca seçim işaretleyicilerini gerek standart medya büyüyecek bir patojen içermeyen dondurulmuş kültür olarak verilmektedir. Deneme için, in vivo gen ekspresyonu veya tumorgenesis optik bir göstergesi olarak hizmet veren lusiferaz gen ifade luc2 4T1 fare meme tümör hücre hattı, kullanacağız. Non-invaziv primer tümörün büyümesini izlemek için luciferase ifade kullanabilirsiniz, ancak onlar da metastatik lezyonlar bulmak ve izlemek için de kullanılabilir. Lusiferaz aktivitesi enjeksiyondan önce ve bunu yapmak için kontrol edilmelidir,% 90 konfluent balon trypsinization hasat ve sonra sayılır. 50.000 hücreler daha sonra bir mikrotiter plate ve seri dilüsyonları tek bir kuyu halinde uygulanmaktadır yapılmaktadır. Luciferase ml&#39;de 150ug kuyulara eklenir ve 2 dakika inkübe. Mikroplak IVIS veya ifade düzeylerini belirlemek için bir ışıldayan plaka okuyucu görüntülü olabilir. Bu hücre hattı, ikinci hücre başına ortalama 6500 fotonlar kadar ifade, ancak ikinci hücre başına ortalama 500 fotonlar üzerindeki herhangi bir ifade seviyesi in vivo olarak başarıyla görüntülü olabilir. Şimdi uygun aktivite hücreleri olduğunu, subkutan enjeksiyon adıma geçebilirsiniz. Tümör optimum algılama kolaylaştırmak amacıyla, immün sistemi baskılanmış bir atimik çıplak fare suşu kullanıyor. Enjeksiyondan önce, hayvanlar derin anestezi için anestezi etkisi vardır. Sonra 100ul PBS içinde 250.000 hücrelerine enjekte edecektir kanadını içine subkutan enjekte edilir. 1 ml şırınga içinde hücreleri Yük ve 26 gauge iğne takın. Forseps bir &quot;çadır&quot; ve tabanda hücreler enjekte etmek için cildi hafifçe kaldırın. Yeni enjekte edilen hücrelerin hemen görüntülü olabilir. Her görüntüleme oturumda kg vücut ağırlığı başına luciferase toplam 150mg sonra periton boşluğuna iki enjeksiyon yoluyla uygulanır. Bu çalışmada, hayvanlar, tutarlı foton akısı sağlamak için luciferase enjeksiyondan sonra 10 dakika görüntülü. Biz bir sonraki adımda bunu göstereceğiz. Luciferase kinetik günden güne değişebilir. Bu model, luciferase enjeksiyondan sonra 10 dakika% 15 değişkenlik aralığında ölçüm sonucu verdi. Deneme için in vivo görüntüleme sistemi, arka inceltilmiş bir şarj bağlantılı aygıt ° C maksimum hassasiyet elde etmek için -90 soğutmalı kullanan IVIS Spektrum kullanacağız. Mutlak kantitatif desteklemek için, sistem aşağı-time sırasında karanlık şarj önlemleri ve başlatma sırasında kendi kendine kalibrasyon çalışır. Görüntüleme başlatmak için, IVIS sistemi başlatmak (tek tıklama) ve deney için görüntüleme parametrelerini ayarlamak. Görüntülü olan hayvanların sayısı için görüş alanını seçin. 5&#39;e kadar hayvan cihazın ayrılmaz bir anestezik manifoldu kullanarak korunabilir. Aşamasında sabit bir 37 ° C hayvanlarda vücut ısısını korumak için. EKG portu stresli işlemleri sırasında hayvan sağlığı izlemek için sağlanır. Görüntü yazılım, maruz kalma süresi, f-stop ve piksel In Living binning hücre hattı ifade düzeyde göre optimize edilebilir. Bu ayarlar kantitatif sonuç etkilemeden bir deney sırasında herhangi bir zamanda değiştirilebilir. IVIS beyaz ışık ve görüntü üzerinde üst üste bir nicel bioluminescent veya floresan sinyali altında hayvan fotografik bir görüntü kazanır. Bioluminescent sinyali saniyede fotonlar ifade ve bir yoğunluk haritası olarak görüntülenir. Görüntü kantitatif etkilemeden görüntü optimum kontrast ve çözünürlük sağlamak için ayarlanır. Hücrelerden Lüminesans bir bölge faiz aracını kullanarak enjeksiyon yerinde ölçülebilir. Ölçüm verileri analiz için kaydedilir veya ihraç edilebilir bir görüntü yakalamak için ilgili tüm deneysel parametreleri ile birlikte tablo görüntülenir Çoklu görüntüleri elde ve deney doğa bağlı olarak saniye veya ay kapsayan boylamsal çalışmalar mukayese edilebilir. Biz zaman sıfır tümör foton akısı ölçmek ve iki haftalık aralıklarla görüntüleme ile 4 hafta boyunca izlemek. Foton akısı tümör biyoparlaklık tümör boyutu ile doğrudan ilişkili lusiferaz ifade canlı hücrelerin sayısı orantılıdır. 5 gün sonrası implantasyon az tümör henüz palpabl değil, ancak hücreleri biyoparlaklık ve aktif büyüyen bir tümör olduğu görüldü sayılabilir olabilir. Bu aşamada biyoparlaklık sinyal çok stronger. Maruz kalma süresi, f-stop ve piksel binning görüntü net ve kamera doyurabilecek değildir ki ayarlanabilir. Bu ölçümler deney daha önce ve daha sonra toplanan bu kıyaslanabilecek kadar IVIS ışık toplama değişiklikleri otomatik olarak dengeler. 7 gün yazılan implantasyon tümörler ölçüm veri zaten 7 gün üretilen ilk kez ve bioluminesans palpabl. 28 gün yazılan implantasyonu, tümörlerin nekrotik hale geliyor ve hücreler ölmeye başlar. Kaliper ölçümü ile tahmin Tümör boyutu kayda değer bir değişiklik değildir, ancak tümör lüminesans hücre ölümü belirten azalacak. Kaliper ölçümleri ve biyoparlaklık ölçüm, insani bir son nokta ulaşana kadar devam edilebilir. Hipoksi veya tedavi rejimleri nedeniyle tümör nekroz, tümör kitlesi azaltmak olmasa bile düşük biyoparlaklık tarafından belirtilecektir.

Kurumsal Hayvan Bakım tarafından onaylanan 060 Hayvan Protokolü ile uyumlu görüntülenmiştir Deneyler makale Journal of üretmek için yapılan tüm deneyler yapıldı sağlamak ve kaliper Yaşam Bilimleri Komitesi (IACUC), Alameda CA Jae Kim, Ph kullan istiyorum D., IACUC Başkanı, kaliper Yaşam Bilimleri.

in vivo bioluminescent imaging of mammary tumors using ivis spectrum

4T1 fare meme tümör hücreleri 15 ila 20 gün içinde palpe edilebilir tümörler oluşturmak üzere alt-kutanöz nu / nu farelere implante edilebilir. Bu xenograftlarının tümör model sistem varsayılan antitümör bileşikler in vivo değerlendirilmesi klinik öncesi için değerlidir. 4T1 hücre hattı yapısal ateşböceği lusiferaz gen (luc2) ifade etmek için tasarlandı. 4T1-luc2 tümörü taşıyan farelerin tümörler IVIS Spektrum gibi hassas optik görüntüleme sistemi kullanılarak takip edilebilir bir görsel ışık sinyali yayan luciferase ile enjekte edilir. Tümör foton akısı ışık yayan hücre sayısı ile doğru orantılıdır ve sinyal tümör büyümesini ve gelişimini izlemek için ölçülebilir. IVIS bioluminescent sinyal ve uzunlamasına çalışmalar mutlak kantitatif ve kantitatif sonuç ödün vermeden birçok ay boyunca sinyal büyüklüğü çeşitli siparişler üzerinde yapılabilir etkinleştirmek için kalibre edilir. , Tümör boyutu, geleneksel fiziksel yollarla palpabl veya ölçülebilir hale gelmeden önce tümör büyümesini biyoparlaklık birkaç gün boyunca izlenebilir. Bu hızlı izleme tümör gelişiminin erken olaylar içgörü sağlamak veya daha kısa deneysel prosedürleri yol. Tümör hücre ölümü ve bioluminescent sinyal bir azalma hipoksi ya da ilaç tedavisi nedeniyle nekroz erken gösterilir. Bu hücre ölümü, fiziksel yollarla ölçülen tümör boyutu bir azalma eşlik olmayabilir. Tümör nekroz erken olayları görmek için yeteneğini terapötik ajanların seçimi ve gelişimi üzerinde önemli bir etkisi vardır. IVIS kullanarak tümör büyümesi nicel görüntüleme hassas kantitatif sağlar ve sonuçları üretmek için deneysel sürecini hızlandırır.

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

Lusiferaz ifade Meme tümör hücrelerini farelerde subkütan implant ve uzunlamasına bir çalışmada non-invaziv tümör büyümesini ve gelişimini izlemek için optik görüntüleme ile görüntülendi.

Meme Tümörleri IVIS Spektrum kullanarak in vivo Bioluminescent Görüntüleme

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 ...

in-vivo-bioluminescent-imaging-of-mammary-tumors-using-ivis-spectrum

Research

JoVE Journal

Biology

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

46.9K Görüntüleme.  Caliper Life Sciences.  中文 

Video: 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.

Meme Görüntüleme Penceresi yoluyla Dendra2 Photoswitching

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

Biyoloji

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.

Derin Kortikal Katmanlar bir micropism kullanarak in vivo Görüntüleme

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.

Mezoskopik Floresans Tomografi için In-vivo Görüntüleme Geliştirme 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.

Meme Epitel Nakli Prosedürü

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.

Görüntüleme Protein-protein etkileşimleri 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.

Gen Yönetmelik Tek molekül Görüntüleme<em&gt; In vivo</em&gt; Dilinim tarafından Cotranslational Aktivasyon (CoTrAC) kullanma

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.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

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.

Whole Sırasının NGS Analiz için bir transpozaz tabanlı Technology tarafından gDNA Zenginleştirme<em&gt; BRCA1</em&gt;<em&gt; BRCA2</emDNA Hasarı Onarım ilgilendim&gt; ve 9 Genler

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.

Bir taranabilir<em&gt; İn Vivo</em&gt; Transjenik biyolojik olarak ışık veren kullanarak Mitokondriyal Modülatörleri için tahlil<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.

sertliği ölçme<em&gt; Ex Vivo</emAtomik Kuvvet Mikroskobu Kullanma&gt; Fare aorta

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