細胞·発生生物学のビデオ撮影機器の部門を使用するためのマーク·ウォズニアック。 ヴァンダービルト大学の細胞イメージング共有リソース。 この作品は、ERPへのAHA助成12PRE10950005、DMBにするDMB、NIH助成金R01 HL037675にAHA助成11GRNT7690040によってサポートされています。 DMBは、心血管研究におけるグラディスP.スタールマンチェアです。

動物に関わるすべての手続きが適切な動物を使用することによって承認されていることを確認し、ボディケア。 1事前に証券バッファを準備します<ol><li> 2Lの株式のCa 2 +フリーのタイロード（ 表1）を準備します。 NaOHでpHを7.4に調整する。 </li><li> 2Lの株式1.2のCa 2 +タイロード（ 表2）を準備します。 NaOHでpHを7.4に調整する。 </li></ol>実験の日に2。、灌流、転送、および消化緩衝液を準備する<ol><li>のCa 2 +フリーのタイロード（ 表3）に150ミリリットルの灌流液を準備し、0.2μmのフィルターを通して濾過する。 </li><li> 35ミリリットルを準備のCa 2 +の無料送迎「バッファーA」「緩衝液B」（ 表5）を含む（ 表4）と25ミリリットルのCa 2 +。 0.2μmのフィルターを介してそれぞれをフィルタリングします。 </li><li> 0の溶液を調製緩衝液Aおよび表6あたりBを混合することにより0.06、0.24、0.6、および1.2のCa 2 +。 </li><li>マウスの体重（BW）に基づいて、25ミリリットルの灌流液（ 表7）に消化酵素を溶解させて25ミリリットルの消化緩衝液を調製する。使用前に0.2μmのフィルターを通してろ過する。 </li></ol> 3実験のセットアップ<ol><li>市販または3ミリリットルの流量を可能にするために蠕動ポンプ、配管、熱交換器コイルを用いて、定流量ランゲンドルフ装置を組み立てる/ 37°Cに加熱した灌流液の分。簡単な概略図がベルら 18によって2011件に設けられている。 </li><li>カニューレから流出が3ml /分の流速で37°Cになるようにランゲンドルフ装置の水浴温度（〜47℃）を循環させる設定。 </li><li>超高純度のI型水100ml、続いて15分間灌流システムを介して70％エタノールを実行する。本稿では、Webを介して実行灌流液5分間のm個の気泡を排除しつつ。 </li><li>目的のメソッドを持つすべての手術器具を滅菌する。 </li><li> 23 Gルアースタブアダプターの先端に、PE-50チューブの短い断片（約3mm）を取り付けることにより、カニューレを作成します。チューブの熱先端が大動脈が配置され、その上リップを作成します。 </li></ol> 4心筋細胞の単離<ol><li>腹腔内注射を介して、0.2ミリリットルのヘパリン溶液（千IU / ml）でマウスを注入する。 </li><li> 5分後、イソフルランでマウスを麻酔。 （強い足のピンチに応答なし）完全に麻酔をかけたときに、70％エタノールで胸をスプレー。胸を開き、すぐに心臓を切除、大動脈、および4℃の灌流液内の場所を傷つけないように注意しながら。把握し、心の下で持ち上げるように湾曲した鉗子を使用してください。これは細かいハサミで心の後ろに添付ファイルをトリミングする領域を作成します。 </li><li>優しく細かい鉗子2対の大動脈の端をつかんで慎重にopeniを引っ張ることにより、心臓にカニューレを挿入カニューレのリップの上大動脈のngの。これは冠状動脈を介して心臓の灌流を阻害するように、カニューレの先端が大動脈弁を過ぎおよび心室にないことを確認してください。 </li><li>すぐにカニューレのリップの上行大動脈の周囲に5-0絹縫合糸のループを接続することによりカニューレに大動脈を固定します。 </li><li>注：4.4を通じて4.2可能な限り迅速に、最高品質の消化のために完了する必要があります手順。 </li><li>注：心はすでに灌流液を流しながらランゲンドルフ装置に取り付けられたカニューレに直接掛けることができます。しかし、解剖顕微鏡下で灌流液の小さなシリンジに取り付けられたカニューレに心臓をぶら下げ最も成功したことが見出されている。これは、冠状動脈のクリアを監視し、心臓が確実にカニューレに取り付けられ、確実に、灌流液の少量を介してプッシュされることを可能にする。カニューレは、その後、灌流液を流水でランゲンドルフに掛けている。 </li><li>灌流2-3分間3ミリリットル/分の流量でのCa 2 +フリーの灌流バッファーで心。 </li><li>消化緩衝液酵素および7-10分間灌流するスイッチ、心は色が柔らかく、軽くなります。 </li><li>注：消化時間は、酵素活性及び心臓の大きさに基づいて調整することができる。 </li><li>心臓が触知弛緩すると、緩衝液A（CA 2 +の無料転送バッファ）で無菌のp60皿にカニューレと場所から心臓を取り出します。 </li><li>心房および大血管を削除します。必要に応じて、右心室を削除します。鉗子を使用すると、小さな断片に心室を分ける。さらに、細胞を分散させるための無菌の5mlホールピペットとピペットを数回。 </li><li> 250μmのナイロンメッシュフィルターに通した50mlコニカルチューブに細胞懸濁液をフィルタリングする。 </li><li>懸濁液は約10分間ペレット化するようにします。 </li><li> 0.06のCa 2 +溶液中にペレットを転送します。細胞を10分間ペレット化するようにします。ほとんどの死細胞が浮遊しながら健康な細胞は、ペレットにします。 </li&gt; <li>上清を吸引し、0.24のCa 2 +の溶液にペレットを移す。 </li><li> 10分間のインキュベーションを繰り返し、吸引し、細胞を1.2 mM溶液になるまで、残りの二つのCa 2 +ソリューションを通じて転送します。この時点で、細胞の大部分は、棒状筋細胞である必要があります。 </li></ol> 5。収縮およびカルシウムトランジェントを測定する<ol><li>注：収縮性とカルシウムトランジェントの両方を同時に測定することができる。 </li><li>を1μg/μlの濃度に無水DMSOに再懸濁することにより、フラ-2 AM溶液を調製する。フラ-2 AM溶液を、-20℃でデシケーター中に保存することができる。 </li><li>負荷1μlのフラ-2 AMで懸濁した細胞の1ミリリットル。細胞は、上清を吸引した後、暗い10分で静置する。細胞は洗浄の間に、10分間、暗所でペレット化することができ、カルシウム2 +タイロード溶液で2回洗浄する。 </li><li>加熱されたPEを可能にする段階文書にカバーガラスを置きrfusion /吸引し、筋細胞フィールド刺激をペーシング電極。倒立顕微鏡に置き、ステージインサートは、油浸対物レンズを使用している場合、レンズへのオイルの滴を追加して、蛍光測定用に設定。 </li><li> 1.2のCa 2 +タイロード液で加熱された重力の灌流を設定し、ステージ上の溶液を37℃に達するようにします。真空吸引を取り付けます。電極を取り付けます。 </li><li>顕微鏡システムの電源を入れ、レシオメトリック蛍光およびセル寸法データ収集ソフトウェアを開きます。 </li><li>瞬間的に灌流の遮断が健康な細胞を沈降させるために、ステージインサートにフラ-2 AM負荷細胞の数滴を追加します。 </li><li>の所望の細胞を配置する対物レンズ40倍を使用してください。健康な細胞は棒状ではなく、自発的に収縮するである必要があります。それは、目に見えて、均一に契約セルは5〜20 Vでペーシングされるべきとき</li><li>注意：生理解析は、高品質の消化を必要とします。それは健康的に見えますが、PAをしない細胞を有することが可能である<html>生理学的な解析のためにうまくCE。消化プロトコルは、高品質、健康的な筋細胞を得るために洗練が必要な場合があります。さらに、変異体​​または疾患の心は、プロトコルへの変更が必要な場合があります。 </li><li>レンズ回転を調整し、所望のセルが画面上に水平に並んで背景が表示されていないされて開口部を変更してください。 </li><li>筋細胞の両端にエッジ検出棒の位置を合わせ、しきい値を調整。均一な筋節での細胞の一部に筋節検出を合わせます。もはやあなたは、このバーを作ることができ、筋節の長さのより正確な数学的モデルは、それが筋節の2集団ではないので、となります。 </li><li>記録前2 Hzから10〜15秒5-20 Vでペース細胞。 </li><li>録音のトレース。周囲の光が最高のカルシウムトランジェント記録用に最小化されるべきである。 </li></ol> 6。分析<ol><li>レシオメトリック蛍光および細胞寸法データ収集ソフトウェアを使用してトレースを分析します。 </li></ol>タイトル &quot;&gt; 7。さらなる研究<ol><li>固定および免疫染色、直接的な薬物の効果を評価する、短期文化、原子間力顕微鏡：このプロトコルは、を含む他の実験さまざまな目的で使用することができ、過剰な細胞を提供する。 </li></ol>

<ol>
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私たちは、開示することは何もない。

質の高い心筋細胞の単離は、練習と最適化のいくつかのレベルが必要です。このプロトコルは、大幅に消化の結果に影響を与えることができます重要なステップが含まれています。プロトコルを実行すると、トラブルシューティングの際にこれらを慎重に検討する必要があります。カニューレ挿入および灌流に対する心臓の除去からの時間は、理想的には5分未満、最小化されるべきである。...

Cardiomyocytes provide the contractile force for the heart. Each myocyte contains organized cytoskeletal and contractile elements essential to contraction. The heart not only contains cardiomyocytes but also fibroblasts, connective tissue, and modified myocytes such as Purkinje fibers. Changes in any or all of these cell types can be seen in heart failure, making determination of the ultimate cause of heart dysfunction difficult. Decreased ventricular contractility of a failing heart is preceded by varying degrees of fibrous tissue build-up and hypertrophy and dysfunction of the cardiomyocytes1. Arrhythmias can be caused by dysfunction in the conducting cells2, fibrosis interrupting cardiac conduction3, or changes in the expression of ion channels in cardiomyocytes1,4. Indeed, these problems often co-exist in a complicated fashion.
Isolating and studying cardiomyocytes allows one to examine contractile and electrical dysfunction of individual myocytes in the context of overall heart function. Techniques to isolate individual cardiac myocytes were developed over 40 years ago in an effort to better study physiology of this &#8216;workhorse&#8217; cell of the heart. Isolation techniques have been improved to increase yield and quality of cells, with the vital development of coronary perfusion via cannulation of the aorta, first performed in 19705. This article describes cardiomyocyte isolation followed by measurement of contractility and calcium transients using ratiometric fluorescence and cell dimensioning data acquisition software. In both genetic and environmental models of heart disease, utilizing this protocol provides key information about cell contractility, sarcomeric contractility and relaxation, calcium transients, and cytoskeletal disruption. This system is often used to test the effect of drugs on isolated cardiomyocytes6,7. We employ the system to compare contractility and calcium transients of cardiomyocytes between wild type mice and mice with a mutation leading to dilated cardiomyopathy.
Studying individual cardiac myocytes has numerous advantages over other commonly used methods to study cardiac anatomy and physiology. Echocardiography and electrocardiography provide information about the contractile function and electrical conduction of the heart overall. Neither of these methods explains the cause or nature of the dysfunction below the level of the whole organ. Measuring contractility of individual cardiomyocytes using edge-detection and sarcomere length algorithms can demonstrate that changes within the contractile cell of the heart are present within global heart dysfunction. Measuring calcium transients demonstrates the influence of changes in ion flux at a cellular level to contractile dysfunction or arrhythmias.
In addition to complementing whole organ studies, this protocol provides cells easily stained for sub-cellular components. It is possible to see cytoskeletal changes by staining tissue sections of the whole heart; however, one is only able to visualize a cross section of cells. A whole cardiomyocyte is thicker than the typical tissue section, and given the arrangement and length of the cardiomyocytes, it is difficult to see whole cells in a single section. Isolated cardiomyocytes can be fixed immediately and stained for a variety of cytoskeletal elements and ion channels. Confocal images can be compiled into full thickness z-stacks of the cell. Additionally, staining isolated cardiac myocytes allows for staining of components that are impossible to visualize in sections, such as the membrane invaginations known as t-tubules.
Cultured cell lines of cardiac myocytes do exist, some of which have similar transcriptional profiles and phenotypic features to cardiac myocytes. Some, such as the HL-1 cell line, even retain some sarcomeric organization and rudimentary contractile ability8. Despite these qualities, cultured cells lack the &#8220;box car&#8221; shape and t-tubule structure of cardiomyocytes and have less sarcomeric organization. These components are essential to the function of cardiomyocytes in vivo. In addition, using cultured cells allows genetic manipulation but not in vivo physiologic manipulation. Isolated cardiomyocytes retain their cellular organization long enough to study but are still easily imaged and even transduced9. 
Isolated cardiomyocytes are extremely versatile, providing a substrate to identically analyze cells from varied experimental conditions. Isolation and physiological analysis of myocytes have been used by countless labs for a variety of experiments including studying the effect of drugs6,7 or small molecules10, environmental stressors11, infection12, illness13, or genetic mutation14,15 on contractility and/or calcium transients and studying the regulation of contractility and calcium release within a cardiomyocyte15-17. With the application of any of these stressors, changes in heart function could be due to any combination of changes in the cardiomyocytes themselves and changes in the surrounding environment of the heart, such as scarring, changes in conductivity, or changes to the extracellular matrix. This is an ideal protocol for answering research questions regarding individual myocyte structure and function in the context of heart failure from any cause.
Although it has countless advantages over other techniques, physiological analysis of isolated cardiomyocytes is not ideal for every research question. Isolation is a terminal procedure, thus cardiomyocytes can only be assessed at one time in the mouse&#8217;s life. This can be partially overcome using cohorts and sacrificing individual mice along the course of illness development or by monitoring the mouse to determine whether it has clinically significant heart failure before isolation. Although this protocol can determine whether cardiomyocyte dysfunction has occurred, one cannot draw the conclusion that this dysfunction is the root cause of heart failure without additional experimental information. It is possible that cardiomyocyte dysfunction itself is secondary to other changes in the heart. Despite these challenges, studying cardiomyocytes offers valuable information in determining the nature of dysfunction in the heart, especially when combined with other experiments. 
The following protocol is adapted from one provided by Dr. Chee Lim, Vanderbilt University. Although contractility and calcium flux data are highly reproducible and useful in comparing hearts with genetic or environmental manipulation, isolation itself remains a technique dependent protocol, requiring optimization. Minimizing the time between removing the heart from the mouse and perfusing the heart via aortic cannulation is essential for a quality digestion. In addition, digestion time and enzyme concentration can be optimized for the highest quality myocytes. Steps likely requiring optimization, along with suggestions for optimization, are noted below.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">NaCl</td>
			<td style="width:221px;">RPI</td>
			<td style="width:159px;">S23020</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">KCl</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">P-3911</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:253px;">MgCl2</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">M-8266</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">HEPES</td>
			<td style="width:221px;">EM Science</td>
			<td style="width:159px;">S320</td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:253px;">NaH2PO4</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">S5011</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:253px;">CaCl2 &#376; 2H2O</td>
			<td style="width:221px;">Fisher</td>
			<td style="width:159px;">C79</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">D-(+)-Glucose</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">G7528</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">2,3-Butanedione-monoxime (BDM)</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">B-0753</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Taurine</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">T-0625</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">A-7030</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Collagenase B</td>
			<td style="width:221px;">Roche Diagnostics</td>
			<td style="width:159px;">1-088-823</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Collagenase D</td>
			<td style="width:221px;">Roche Diagnostics</td>
			<td style="width:159px;">1-088-882</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Protease XIV</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">P-5147</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Heparin</td>
			<td style="width:221px;">APP Pharmaceuticals, LLC</td>
			<td style="width:159px;">401586D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Isofluorane</td>
			<td style="width:221px;">Butler Schein</td>
			<td style="width:159px;">11695-6776-2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Fura-2 AM</td>
			<td style="width:221px;">Teflabs</td>
			<td style="width:159px;">103</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">DMSO &#8211; anhydrous</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">276855</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:253px;">IonOptix cell pacing and fluorescent analysis system</td>
			<td style="width:221px;">IonOptix</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">250 um nylon mesh filter</td>
			<td style="width:221px;">Sefar America</td>
			<td style="width:159px;">Lab Pak 03-250/50</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">23G leur-stub adaptor</td>
			<td style="width:221px;">Becton Dickinson</td>
			<td style="width:159px;">1482619E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">PE-50 tubing</td>
			<td style="width:221px;">Becton Dickinson</td>
			<td style="width:159px;">427410</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and physiological analysis of mouse cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

（ 図2）の収縮性と過渡追跡が収集されると、データは容易に適切なソフトウェアを用いて分析される。全体トレースまたはその一部（ 図3）を平均化することができる。収縮性は、さまざまな方法で分析することができる。収縮機能のために、人はショートニングピークまでの時間、および収縮速度を短縮率と収縮の大きさ、または収縮の速度を評価することがで?...

Watch this Scientific Journal Video about Isolation and Physiological Analysis of Mouse Cardiomyocytes at JoVE.com

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

マウス心筋細胞の単離と生理解析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

isolation-and-physiological-analysis-of-mouse-cardiomyocytes

Research

JoVE Journal

Biology

22.8K ビュー.  Vanderbilt University.  Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause. 

ビデオ: Isolation and Physiological Analysis of Mouse Cardiomyocytes

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

成体マウスから海馬歯状回の解剖

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成体マウスの心筋細胞の単一細胞転写プロファイリング

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

マウス心房と心室の心筋細胞における分離とKvチャンネル録音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

新生仔マウス心筋細胞の単離および培養

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

細胞シグナリングのための成体マウスの心筋細胞の単離と文化<em&gt; in vitroで</em&gt;心肥大

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

培養細胞およびマウス組織から単離とミトコンドリアの機能解析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

成体マウスの心筋細胞の単離、培養および形質導入

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

成体ラット心臓からの高品質心筋細胞、内皮細胞および線維芽細胞の同時単離

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

そのままに、全体のマウス乳腺の腺の形態と細胞外マトリックス式の分析のための分離

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...