Name: mammalian cell division in 3d matrices via quantitative confocal reflection microscopy
Uploaded: 2017-11-29T14:00:00
Description: Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

この作品は、NIH によって支えられた R01CA174388 と U54CA143868 を与えます。著者は魏堂陳のサポートのためのジョンズ ・ ホプキンス大学からプラ賞を認めたいと思います。この資料は、国立科学財団大学院研究奨学金助成金第 1232825 の下に支えられて仕事に基づいています。

提供されるプロトコルのホームウッド制度検討委員会 (HIRB) のガイドラインに従います。
1. 細胞分裂のマーカーとしての H2B mCherry 安定式
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(HEK 293 t) から人間の萌芽期の腎臓 293 t レンチ ウイルス粒子の生成細胞
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<li>細胞培養培地 (ダルベッコ変更イーグル培地 (DMEM) 含む高グルコース (4.5 g/L)、ピルビン酸ナトリウム 10% 胎仔ウシ血清 (FBS)、1% の 10 の...

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ablation+or+inhibition+of+stromal+matrix+metalloproteinase-9+on+lung+metastasis+in+a+breast+cancer+model+is+dependent+on+genetic+background.">Effect of ablation or inhibition of stromal matrix metalloproteinase-9 on lung metastasis in a breast cancer model is dependent on genetic background.</a> Cancer Res. 68 (15), 6251-6259 (2008).</li><li>Knox, J. J., Hotte, S. J., Kollmannsberger, C., Winquist, E., Fisher, B., Eisenhauer, E. 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3 D の細胞分裂の前の研究は、実験的限界と技術的な課題18,19による効率的でした。3 D コラーゲン マトリックス中の哺乳類の細胞分裂の学習の効率化のための重要なステップは、: (1); 細胞分裂マーカーの蛍光標識定款(2) 細胞分裂; の同期(3) 生細胞イメージング、共焦点反射の時間分解顕微鏡と定量的イメージング解析を用いた 3 D マトリックス部?...

細胞分裂は、うち規制組織や臓器の開発において重要な役割を果たしている細胞の生命の重大なイベントです。異常な細胞分裂、自然な遺伝的変異、ヒトの老化プロセス、およびがん1,2,3,4、5の進行に関与しています。正常細胞と比較して腫瘍細胞の増殖の増加率は、癌細胞の挙動は腫瘍の種類と患者の中でもかなり異質であるという事実にもかかわらずの特徴のひとつです。前臨床試験の有望な結果にもかかわらずいくつかの新たに開発された抗がん剤薬が臨床試験6,7,8,9,10 に有効であるには表示されません。、11。実験および臨床モデルの妥当性は考慮すべき。三次元 (3 D) マトリックス、線維芽細胞とコラーゲンが豊富な 3 D 結合組織、線維肉腫細胞 3 D 間質の細胞外マトリックス (ECM) の転移性癌細胞などの正常な哺乳類やがん細胞の多くの種類を分けます。ただし、二次元 (2 D) 基板上に培養した細胞を実験哺乳類の細胞分裂との試金の大半を行った。組織, 機械的性質および両方正常と病理組織12,13,14,の 3 D の ECM の生化学的なシグナルを設計された 3 D マトリックスが要約より15,16,17。
どの哺乳類の細胞分裂の研究の規制環境が生理学的な関連性や治療の意義18,19にもかかわらず主未踏のまま 3 D で。技術的な問題と 3 D マトリックスの細胞分裂の勉強に関連する実験課題がある可能性があります。細胞分裂は、全体の細胞周期20のほんの時間を構成します。前の仕事は人間の乳房腺 MCF 7、ひと骨肉腫 U2OS、人間の肝臓などの多くの哺乳類細胞の増殖率を示す 2D 基板21、相手と比較して 3 D の行列の hepg2 細胞がはるかに低い 22。さらに、3 D マトリックスに埋め込まれた細胞は、フォーカスのうち住セルイメージ投射の間に移動します。これらすべての要因は、イメージング技術を用いた三次元培養における細胞分裂イベントのキャプチャの非常に低い効率に貢献します。
ECM と細胞間相互作用は、細胞分裂の調節に重要な役割を再生します。ここでは、3 D コラーゲン マトリックス中の哺乳類の細胞分裂を効率的に勉強する方法をについて説明します。メソッドは、細胞、細胞分裂、同期生細胞イメージング、共焦点反射の時間分解顕微鏡を使用して 3 D の行列の部イベントの監視に分裂のマーカーの取り込みを含むと定量的画像解析。蛍光標識ヒストン H2B が最初に導入された細胞に細胞分裂を区別し、中間期の細胞マーカーとして。その後、セルは、機械的振り払いテクニックが続きます、チミジンをブロックし、ノコダゾール治療の組み合わせを使用して同期されます。3 D コラーゲン マトリックスに同期された細胞が直接カプセル化されます。複数のセルの細胞分裂イベントは、効率的に低倍率のタイムラプス住セルイメージ投射を使用して監視されます。細胞マトリックスの相互作用の指標となるコラーゲン線維の変形は、高倍率で反射共焦点の顕微鏡検査を使用して監視されます。
監視し前に、中、後 2 つの転移性癌細胞株、ひと浸潤性膵癌 MDA MB 231、3 D コラーゲンのひと線維肉腫 HT1080 細胞の有糸分裂細胞マトリックスの相互作用を定量化するこのテクニックを使いました以前行列19。紹介した方法は、3 D 環境と細胞マトリックスの相互作用で両方の哺乳類の細胞分裂の研究に効率的で一般的なアプローチを提供します。MDA MB 231 細胞ラインは、ホワイト ペーパーで例として使用されます。このプロトコルは正常組織および病気の開発の分子基盤に新しい洞察力を提供し、新規診断と治療的アプローチの設計もできます。

<table>
	<tbody>
		<tr>
			<td>Human embryonic kidney 293T</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>Physical Sciences Oncology Center, NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>ThermoFisher Scientific</td>
			<td>12100-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100X</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene HD</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life technologies</td>
			<td>11668-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid encoding H2B-mCherry in a lentiviral vector</td>
			<td>Addgene</td>
			<td>plasmid 21217</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>GibcoBRL</td>
			<td>11810-025</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>113375-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>J.T. Bake</td>
			<td>3722-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV syringe filter unit, 0.45-&#956;m, PVDF, 33 mm</td>
			<td>Millipore</td>
			<td>SLHVM33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon TE2000E epifluorescence microscope</td>
			<td>Nikon</td>
			<td>TE2000E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade 1K CCD camera</td>
			<td>Roper Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements AR imaging software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1 confocal microscope</td>
			<td>Nikon</td>
			<td>A1</td>
			<td></td>
		</tr>
	</tbody>
</table>

mammalian cell division in 3d matrices via quantitative confocal reflection microscopy

どの哺乳類の細胞分裂の研究の規制 3 d 環境生理学的な関連性や治療の意義にもかかわらず主として未踏のままです。探査の欠如の原因は、実験的限界と非効率的な三次元培養における細胞分裂の研究をレンダリング技術の課題です。ここでは、哺乳類の細胞分裂と細胞マトリックス 3 D コラーゲン マトリックス相互作用を効率的に勉強するイメージング ベースの方法をについて説明します。蛍光 H2B の付いたセルは、機械的振り払いテクニックが続きます、チミジンをブロックし、ノコダゾール治療の組み合わせを使用して同期されます。同期セルに 3 D コラーゲン マトリックスに埋め込まれています。細胞分裂は細胞の顕微鏡を使用して監視されます。コラーゲン線維の変形中に、細胞分裂、細胞マトリックスの相互作用の指標である後の監視し、量的共焦点反射の顕微鏡検査を使用して量を示されます。メソッドは、哺乳類の細胞分裂と細胞マトリックス相互作用の生理学的関連性の高い 3 D 環境での勉強を効率的かつ一般的なアプローチを提供します。このアプローチは、正常組織および病気の開発の分子基盤に新しい洞察力を提供するだけでなく、新規診断と治療的アプローチの設計ができます。

この記事の目標は、3 D マトリックスは、哺乳類の細胞分裂過程を研究し、中と細胞分裂後にセルと 3 D の細胞外マトリックスの相互作用を定量化するイメージング ベースの方法を提示することです。細胞分裂の観察を容易にするには、我々 はレンチ ウイルス伝達を使用して MDA MB 231 セルに H2B mCherry を組み込みます。蛍光タンパク質を用いた共役 H2B は、間期の細胞?...

Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

このプロトコルは細胞分裂の同期を統合することで効率的に 3 D コラーゲン マトリックス中の哺乳類の細胞分裂を研究生細胞イメージング、共焦点反射の時間分解顕微鏡を使用して 3 D の行列の部イベントの監視定量画像解析。

哺乳類の細胞分裂量的共焦点反射顕微鏡を介して 3 D 行列

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

The overall goal of this procedure is to study mammalian cell division and cell matrix interactions in a physiologically relevant 3D environment. The main advantage of this technique is that it provides an efficient and general approach to study mammalian cell division and cell matrix interactions. This method can help answer key questions in cell biology such as the molecular basis of the development of normal and disease tissue.A day prior to transfection, plate five million HEK-293 T-cells in a 100 milliliter cell culture dish in 10 milliliters of normal cell culture medium. Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day just before starting transfections, remove a vial of the transfection reagent from four degrees Celsius and leave it out to reach room temperature.Then to start, add one milliliter of reduced serum medium to a sterile microfuge tube. To this, add specified amounts of lentiviral vector plasmids used for transfection and mix by pipetting. Incubate the tube at room temperature for five minutes.After five minutes, add 48 microliters of the transfection reagent into this tube and mix gently by pipetting. Subsequently, incubate this mixture at room temperature for at least 15 minutes. Once the incubation is complete, remove the plate with HEK-293 T-cells from the incubator.Add the prepared DNA transfection reagent mixture drop wise onto the cells and gently swirl the plate to ensure even distribution in the plate. Place the plate back into the incubator. Approximately six hours after transfection, remove the plate and aspirate the medium.Add 10 milliliters of fresh culture medium and transfer the plate back into the incubator. At multiple time points, 24, 48, and 72 hours post-transfection, harvest the culture medium containing the lentiviral particles in separate tubes. Next, filter the harvested culture medium through a sterile 0.45 micron filter to remove cellular debris.Replace with fresh culture medium following each harvest. The filtrate containing the lentiviral particles can be used for transduction right away or can be stored at minus 80 degrees Celsius. Plate human breast carcinoma cells MDA-MB-231 in a 35 millimeter culture dish in normal culture medium.Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day, aspirate the culture medium from the plate and add one milliliter of the lentiviral particles together with one milliliter of fresh culture medium. Incubate the cells in the incubator for about 16 hours.After the incubation with viral particles is complete, remove the plate to aspirate the medium. Then add two milliliters of fresh medium and transfer back to the incubator for 24 to 72 hours. Check the expression of H2B-mCherry in these transduced cells using an epifluorescence microscope.To start the synchronization experiments, first plate MDA-MB-231 cells expressing H2B-mCherry at 50 to 60%confluency in a 24-well plate. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, replace the culture medium with 0.5 milliliters of medium containing two millimolar Thymidine.Leave the plate in the incubator for 24 hours. Once the incubation is complete, wash the cells three times with PBS to release them from the Thymidine treatment. Then leave the cells in 0.5 milliliters of normal culture medium for five hours in the incubator.After this, to block these cells in mitosis, add Nocodazole to the culture medium. Leave the cells in the incubator for 12 hours. After the Nocodazole treatment is complete, take out the plate and using an orbital shaker, shake the cells for 45 seconds to one minute to release mitotic cells.Subsequently, transfer the medium containing the extracted mitotic cells into a new centrifuge tube. Then add 0.5 milliliters of fresh medium to each well. After, repeat the shaking and extraction of cells two more times.Pool the total collected medium and then centrifuge it to pellet the mitotic cells. After the spin, resuspend the cell pellet in 0.25 milliliters of fresh cell culture medium. Using a hemocytometer, count the number of cells.At the start of this step, prepare 50 milliliters of 10x DMEM solution and filter sterilize it. Next, determine the volumes of all the components that will be used to make the 3D collagen matrix as specified in the text protocol. Pre-chill all these components on ice to slow down the gelation of collagen.Now, to a pre-chilled 1.5 milliliter centrifuge tube, add the required numbers of cells in medium then 10x DMEM, FBS, deionized water, collagen, and finally sodium hydroxide. Mix carefully using a one milliliter pipette. Once the solution is well mixed, add 500 microliters of the mixture into each well of the 24-well plate.Place the 24-well plate in a 37 degree Celsius incubator. After 30 minutes, take out the plate and add 500 microliters of pre-warmed culture medium on top of the gel. Prior to imaging, turn on the live cell units of the phase contrast and confocal microscopes.To capture dividing cells, place the 24-well plate containing the cells embedded in collagen matrices into the live cell unit of the fluorescence microscope. Using the 10X objective, find the bottom of the plate and then move it up until the microscope focuses at about 500 micrometers from the bottom of the plate. Move the stage around to find cells in different stages of mitosis and select multiple positions for imaging.Set up the timelapse experiment with two-minute intervals. Next, to image the collagen network, configure the confocal microscope to capture only reflected light from the 488 nanometer laser. Then place the plate containing cells in collagen matrices into the live cell unit of this microscope.Find the bottom of the plate and then move the objective lens up until it focuses at about 100 micrometers from the bottom of the plate. Move the stage around to find cells and select multiple positions for imaging. Set up the timelapse experiment with five-minute intervals.Fluorescent images of a dividing H2B-mCherry expressing MDA-MB-231 cell embedded in collagen matrix are shown here. Different phases of mitosis are readily observed as expected. The distribution of collagen fibers in white visualized simultaneously using confocal reflection microscopy changes very little during the course of mitosis.Cell synchronized G2-M phase and embedded in a collagen matrix complete cell division in the first two hours while control asynchronous cells divide randomly. Quantification of the distribution of collagen fibers during interphase and mitotic phases suggest that matrix deformation is minimal during cell division and higher in interphase cells. Once mastered, this technique can be completed in seven days starting from generating the stable cell line to analyzing the video cell division in a 3D matrix.After watching this video, you hopefully have a good understanding of how to synchronize and monitor mammalian cell division in 3D matrices using live cell imaging and how to determine matrix deformation using confocal reflection microscopy and quantitative imaging analysis techniques.

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Cell-Matrix Interactions

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Video-rate Scanning Confocal Microscopy and Microendoscopy

ビデオレートでスキャニング共焦点顕微鏡とMicroendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

生物工学

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

全内部反射蛍光顕微鏡を用いた微生物のCortex組織とダイナミクスの可視化、

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

可変角度全内部反射蛍光顕微鏡（VA-TIRFM）の際に、細胞接着のナノトポロジー

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

ダイナミックな相互作用の3次元構造解析のための相関顕微鏡

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

定量的な光学顕微鏡：細胞生物物理の測定は、標準的な光学顕微鏡で特徴

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

パラメータ化拡張期充満フォーマリズムを経由Transmitral流れの運動学的モデルベースの​​解析によるグローバル拡張期機能の定量化

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

線状インベーゾームの形成および活性を研究するための2Dおよび3Dマトリックス

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

単一細胞のゲノムの配列のための超高スループット マイクロ流体プラットフォーム

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

剛体の埋め込み固定し、ステンド グラス、全体、マイクロ コンピューター断層撮影によってセクション-無料の 3 D の組織学のためのミリ波スケール標本

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Novel Process for 3D Printing Decellularized Matrices

3 D プリントのための新規プロセス脱行列

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...