ルシフェラーゼを発現する癌細胞株の多種多様なマウスモデルにおける前臨床研究に使用することができます。 これらの細胞は容易に選択マーカーを必要とせず、標準のメディアが大きくなると思われる無菌凍結培養物として提供されています。 私たちの実験では、我々はin vivoでの遺伝子発現やtumorgenesisの光学指標となるルシフェラーゼ遺伝子を発現4T1 - luc2マウス乳腺腫瘍細胞株を、使用します。我々は、非侵襲的に原発腫瘍の成長を追跡するためにルシフェラーゼ発現を使用しますが、彼らはまた、転移性病変を検索し、監視するために使用することができます。 ルシフェラーゼ活性は、注射する前に検証する必要があります、そしてこれを実行するためには、90％コンフルエントフラスコをトリプシン処理によって回収してからカウントされます。 50,000個の細胞は、マイクロタイタープレートの1ウェルに分注しており、連続希釈が行われる。ルシフェリン、1m​​l当たり150ugでウェルに添加し、2分間インキュベートする。マイクロプレートは、IVISまたは発現レベルを決定するために発光プレートリーダーで画像化することができます。この細胞株は、毎秒のセルあたり6500光子まで表現し、1秒あたりのセル当たり500光子上記のいずれかの発現レベルは、in vivoで正常に画像化することができます。 今我々は最適な活性の細胞を持っていることを、我々は、皮下注射の手順に進むことができます。 腫瘍の最適な検出を容易にするために、我々は胸腺免疫不全ヌードマウス系統を使用しています。注射の前に、動物は深い麻酔のための効果に麻酔する。 次に我々は100ulのPBSに250,000細胞まで注入されることは脇腹に皮下注射されています。 1mlの注射器で細胞をロードし、26ゲージの針を取り付けます。鉗子は&quot;テント&quot;を作り、基地の細胞を注入することでお肌を持ち上げます。新たに注入された細胞は、すぐにイメージングすることができます。各イメージングセッションでは体重kg当たりルシフェリンの150mgの合計は、腹腔内に2回の注射により投与される。 本研究では、動物が一貫した光子束を確保するためにルシフェリンの投与後10分をイメージされています。我々は、次のステップであなたにこれを紹介します。ルシフェリン反応速度は日によって異なることができる。この特定のモデルでは、ルシフェリンの投与後10分を測定することで15％のばらつきの範囲内の結果を与えた。 私たちの実験のために我々は、in vivoイメージングシステムでIVISスペクトラムを使用します裏面入射電荷結合素子を使用して° C最高感度を達成するために-90に冷却。絶対定量をサポートするために、システムはダウンタイム中に暗い電荷を測定し、初期化時にセルフキャリブレーションを実行します。イメージングを開始するには、IVISシステムを初期化する（ワンクリック）と実験用イメージングパラメータを設定します。 撮像される動物の数のビューのフィールドを選択します。最大5匹の動物に不可欠な麻酔のマニホールドを使用して楽器に維持することができる。ステージでは、° C、動物の体温を維持するために一定の37℃です。 EKGポートは、ストレスの多い処置中に動物の健康を監視するために提供されています。 画像ソフトをリビングでは、露光時間、F - stopとピクセルビニングは、細胞株の発現レベルに基づいて最適化することができます。これらの設定は、定量的な結果に影響を与えることなく、実験中にいつでも変更することができます。 IVISは、白色光と画像の上に重ねている定量的な生物発光または蛍光シグナル、下の動物の写真画像を取得。 生物発光シグナルは、秒あたりの光子で表され、強度マップとして表示されます。画像表示は、定量に影響を与えることなく、画像の最適なコントラストと分解能を提供するように調整されます。 細胞からの発光は、関心のツールの域を使用して、注射の部位で測定することができます。測定データは、分析のために保存またはエクスポートすることができます画像のキャプチャに関連するすべての実験パラメータと一緒にテーブルに表示されます複数の画像を取得し、実験の性質に応じて数秒または数か月をカバーする長期的な研究に比較することができます。我々は、隔週の間隔で撮影して4週間の時間がゼロとモニターで腫瘍から光子束を測定します。 腫瘍からのフォトンフラックスは、生物発光は、腫瘍の大きさと直接相関するので、ルシフェラーゼを発現する生細胞の数に比例します。 5日後の移植で腫瘍はまだ明白ではないが、細胞は生物発光によって定量化することができると腫瘍が積極的に成長しているように見られている。この段階での生物発光シグナルは非常にstrongeですR.露光時間は、F - stopとピクセルビニングは、画像が鮮明になるとカメラが飽和しないように調整することができます。これらの測定は実験の前半​​で、後に収集したものに比較することができるように、IVISは、自動的に集光の変化を補正。 7日間でポスト移植腫瘍は、測定値が既にデータの7日間を生成したのは今回が初めてと生物発光のための触知です。 28日のポストの移植で、腫瘍は壊死性になりつつあると細胞が死ぬことを始める。キャリパー測定により推定された腫瘍のサイズがかなり変更されませんが、腫瘍からの発光は、細胞死を示す減少します。 人道的エンドポイントに到達するまで、キャリパーの測定と生物発光の測定を継続することができます。低酸素症または治療体制に起因する腫瘍壊死は、それらが腫瘍塊を削減していない場合でも減少生物発光によって示されます。

私は可視化実験の記事のジャーナルを生成するために実行されるすべての実験は動物実験によって承認された動物のプロトコル060に準拠して行われたことを保証し、キャリパーライフサイエンスにおける委員会（IACUC）、アラメダカリフォルニア州在金は、Phを使用したいと思います。D.、IACUCチェア、キャリパーライフサイエンス。

in vivo bioluminescent imaging of mammary tumors using ivis spectrum

4T1マウス乳癌細胞は、15〜20日に触知腫瘍を形成するために、nu / nuマウスに皮下移植することができる。この異種移植腫瘍モデル系では、推定上の抗腫瘍化合物の in vivo評価における前臨床試験のために貴重です。 4T1細胞株は、構成的にホタルルシフェラーゼ遺伝子（luc2）を発現するように設計されています。 4T1 - luc2腫瘍を持つマウスは、腫瘍がIVISスペクトラムのように敏感な光学イメージングシステムを使用して監視することができる視覚的な光信号を発するルシフェリンを注入されている場合。腫瘍からの光子束は、発光細胞の数に比例し、信号が腫瘍の成長と発展を監視するために測定することができます。 IVISは、何ヶ月にわたって実施し、信号の数桁以上の定量的な結果を損なうことなく可能な生物発光信号と縦断的研究の絶対定量を可能にするために較正されています。 腫瘍の大きさは従来の物理的手段によって触知または測定可能になる前に腫瘍の成長は、生物発光で数日間監視することができます。この急速なモニタリングは、腫瘍の開発の初期段階でのイベントへの洞察を提供したり、短い実験手順につながることができます。 低酸素症または薬物治療による腫瘍細胞死と壊死を​​早期に生物発光信号の減少によって示されます。この細胞死は、物理的手段によって測定された腫瘍の大きさの減少を伴うされていない可能性があります。腫瘍壊死の初期のイベントを表示する機能は、治療薬の選択と開発に大きな影響を与えます。 IVISを用いて腫瘍の増殖の定量的イメージングは​​、正確な定量を提供し、結果を生成する実験的なプロセスを加速させます。

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

ルシフェラーゼを発現する乳腺腫瘍細胞をマウスに皮下移植し、縦断的研究で、非侵襲的に腫瘍の成長と発展を監視する光イメージングを用いて可視化されています。

IVISスペクトラムを用いた乳腺腫瘍の in vivo生物発光イメージングに

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

46.8K ビュー.  Caliper Life Sciences.  中文 

ビデオ: 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.

乳腺イメージングの窓から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 ...

生物学

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.

マイクロプリズムを使ってDeep皮質層のin vivoイメージング

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.

のためのメゾスコピック蛍光トモグラフィー生体内</em開発の>イメージングショウジョウバエ</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.

乳腺上皮移植の手順

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.

イメージングタンパク質間相互作用 in vivoで</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.

遺伝子調節の1分子蛍光イメージング<emインビボ&gt;</em&gt;開裂により同時翻訳アクティベーションの使用（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.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

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.

の全配列のNGS解析のためのトランスポゼースベースの技術によるgDNAのエンリッチメント<em&gt; BRCA1</em&gt;<em&gt; BRCA2</em&gt;、およびDNA損傷修復に関与する遺伝子9

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.

スクリーニング可能<em&gt;インビボ</em&gt;トランスジェニック生物発光を使用したミトコンドリアモジュレーターについてのアッセイ<em&gt;線虫（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.

の剛性を測定します<em&gt; ex vivoで</em原子間力顕微鏡を使用&gt;マウス大動脈

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