This work was supported by National Eye Institute Grant R01 EY017724 (VMF) and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant K99 AR066534 (DSG).

全ての動物手順は、国立衛生研究所によってとスクリップス研究所の施設内動物管理使用委員会によって承認されたプロトコルの下で実験動物の管理と使用に関するガイドの推奨に従って行いました。 1.レンズ解剖<ol><li>国立衛生研究所「実験動物の管理と使用に関する指針」および承認された機関の動物使用プロトコルの推奨事項に従ってマウスを安楽死させます。 </li><li>湾曲した鉗子を用いて、マウスから眼を摘出。ソケットから目を持って来るためにピンセットで目の周りの組織を押し下げ、その後、ピンセットでソケットから目を摘み取ります。解剖皿に新鮮な1×リン酸緩衝生理食塩水（PBS）に目を移します。 </li><li>できるだけ眼球に近い視神経を切断。ゆっくりと慎重に穴wherを通して眼球内に微細なストレートピンセットを挿入電子視神経は後方を終了します。 </li><li>慎重に、角膜の端に後方から眼球内にハサミで切開を行います。げっ歯類のレンズは、眼の〜30％を占めます。慎重にこれらの切開を行い、レンズの損傷を防ぐために、眼の中に深すぎるピンセットやハサミを入れないでください。 </li><li>少なくとも半分眼球の周りに角膜と強膜との間の接合部に沿ってカットします。 </li><li>静かにステップ1.4と1.5で作られた開口部を通って眼からレンズを除去するために、角膜を押します。 </li><li>慎重に、まだレンズに装着されている任意の大きな破片を除去するために先端の細いストレートピンセットを使用してください。視覚的に剛性の測定に進む前に、いかなる損害についてレンズを検査します。 </li></ol> 2.剛性の測定<ol><li>化学天秤を使用して、同じボックスからの少なくとも10カバースリップを計量。カバースリップの平均重量を探します。一貫性を保つため、すべての実験のためのカバースリップの同じボックスを使用します。プリウェット実験を開始する前に、少なくとも2時間、室温で1×PBS中でカバースリップと直角ミラー。 </li><li> 1×PBSの75ミリリットル- 65で（ 図1を参照）測定室を埋めます。測定室は、社内の機械工場によってプレキシグラスから作られた、チャンバ内のディボットは、適切なドリルビットで所望の深さに設定ドリルプレスによって作られました。レンズは、機械的試験の期間中、室温で1×PBS中に透明なままです。 </li></ol><img alt="図1" src="/files/ftp_upload/53986/53986fig1.jpg" /> 図1：剛性測定チャンバー異なる深さや形状のディボットの様々なカスタムメイドの剛性測定室の寸法を示す写真。 200ミクロンまたは300ミクロンの深（黄色矢印）あるラウンドのディボットは、マウスのレンズ上の測定に使用されています。ディボットは、直径2ミリメートルと〜13です-チャンバの端から14ミリメートル。 <a href="https://www.jove.com/files/ftp_upload/53986/53986fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <ol start="3"><li>レンズを保持するために使用されるディボットから一定の距離でチャンバ内に直角ミラーを配置します。ミラーは、実験中に動かないことを確認してください。 </li><li>転送は、鉗子または湾曲鉗子を押収して慎重に測定室にレンズを解剖しました。 </li><li>真上からアンロードされたレンズのトップビュー画像を撮影します。顕微鏡（下）と左右の光ファイバ光源を解剖からの照明を用いて30倍の倍率でのマウスレンズの写真を撮ります。最大光強度の80％まで光ファイバ電源を設定します。必要に応じて、周囲の照明、ユーザの嗜好や画質に基づいて電源出力を調整します。 </li><li> RIを通して見ることができる無負荷レンズのサイドビュー画像を撮りますGHT-角度ミラー。カメラが校正されていない場合は、フォーカスのミラーエッジの写真を撮ります。ミラーのエッジを5mmの長さであり、この測定は、後述のピクセル/ mmで決定し、画像中のスケールバーとして機能するように使用することができます。 </li><li>場所ディボットにレンズ、およびレンズがディボットにしっかりとまっすぐに装着されていることを確認します。ロードする前にレンズの写真を撮ります。レンズは、その前方または後方のポール上のディボットで休んでする必要があります。 </li><li>レンズの上にそっと1カバーガラスを置きます。クリープを可能にするために2分待ってから、ロードされたレンズの別のサイドビュー写真を撮ります。 </li><li> 10カバースリップの合計が適用されるまで、ステップ2.8のようにカバースリップを追加し、ステップ2.8のように各カバースリップを添加した後、サイドビューの写真撮影を続行。 </li><li>すべてのカバースリップを削除します。 2分待ってから、すべてのカバースリップを除去した後、レンズ（内側と外側ディボットの）のサイドビュー写真を撮ります。 </li></ol> 3.レンズ核の測定<ol><li> determへレンズ核サイズINE、1×PBSで満たされたきれいなペトリ皿にレンズを移動させます。 </li><li>優しく細かいストレートピンセットを用いてレンズをデカプセル化。 </li><li>手袋をした指の間レンズを転がすことにより、皮質線維細胞脱落。残りのレンズ核は、ハード大理石のように感じるでしょう。生後1ヶ月から始まる大人のレンズに核を単離するために、この手順を使用します。単離された核が剛体であるため、レンズ核のさらなる機械的試験は、この記載された方法を用いて行うことができません。 </li><li>優しくペトリ皿に1×PBS中の水晶体核をすすぎます。 </li><li>バック（ないディボット中）測定室に水晶体核を配置し、直角ミラーを介してレンズ核の画像を取ります。 </li></ol><img alt="図2" src="/files/ftp_upload/53986/53986fig2.jpg" /> 図2：元のカバーガラスにより圧縮されたマウスのレンズ （A）回路図および（B）の写真1×PBSで満たされた測定室で200μmの深いディボットに2ヶ月齢のマウスのレンズを示すperimentalセットアップ。直角ミラーと解剖顕微鏡に搭載のデジタルカメラは、カバーガラスにより圧縮時のレンズの画像を収集するために使用されました。 （C）連続したカバースリップの数を増加させることにより、圧縮2ヶ月齢の野生型レンズのサジタルビューの写真は、軸方向および赤道直径を測定し、カバーガラスベースの圧縮テスト中に軸方向および赤道株を計算するための生データを提供しました。レンズの反射は、時々（1カバースリップの画像の中で最も明瞭に見える）カバーガラスに見ることができます。測定を行うときは、反射を無視し、レンズの頂点に測定します。 （D）2ヶ月齢の野生型レンズ圧縮後と分離された水晶体核の矢状景色の写真。圧縮後のレンズおよび単離された核はディボットの外に座っています。スケールバー、1ミリメートル。この図は、Gokhから変更されていますら 、に。PLoSの1、2012 19。 <a href="https://www.jove.com/files/ftp_upload/53986/53986fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 4.画像解析<ol><li>ロードの前およびImageJのまたは類似のソフトウェアを使用して、各負荷ステップの後にレンズの赤道と軸方向の直径を測定します。各レンズ核の直径を測定します。任意の方向での測定は19,21を十分ですので、レンズ核はほぼ球形です。 </li><li>使用ディボットの深さを追加することで、レンズの軸方向の直径を修正してください。測定室では、ディボットは、レンズの軸方向の厚さの200ミクロン（2ヶ月齢マウスレンズ）または300ミクロン（4ヶ月齢と8ヶ月齢のマウスのレンズを）隠さ。 </li><li>式を用いて、レンズ径の測定値から軸方向および赤道株を計算し、ε=（D - D 0）εが株である/ D 0、dは、軸方向またはEであり、quatorial与えられた負荷時の直径であり、d 0は、ゼロ負荷で対応する軸方向または赤道直径です。 </li><li> （mg単位）かかる負荷の関数として軸方向および赤道株をプロットします。 </li><li>軸、赤道と核の直径をプロットします。赤道直径で軸方向の直径を除算することにより、レンズのアスペクト比を計算し、プロットします。 </li><li>計算し、プロット式を用いてレンズのボリュームを、R eは赤道半径であり、r Aはステップ2.6で撮影した画像から測定軸方向の半径であるボリューム= 4/3×π×R E 2×R A、。この式は、レンズが扁球（楕円）1,22であると仮定します。 </li><li>ステップ3.5で撮影した画像から測定されるようにR Nはレンズ核の半径であるR N 3、×π×計算し、プロットする式を用いた核ボリュームを、ボリューム= 4/3。この式は、レンズ核私を前提としていSA球19,21。 </li><li>レンズボリュームへの核体積の比として核画分を計算し、プロットします。 </li></ol>

<ol>
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著者らは、開示することは何もありません。

レンズの剛性を測定するために、このメソッドを使用する際には、いくつかの重要な考慮事項があります。室（θ）の底面に対して - （8.5°8）まず、カバーガラスを少し斜めの角度でレンズに適用されます。これは非常に小さな負荷のコンポーネント赤道ではなく、軸方向に適用されます。罪θ≈0.1 19しかし、この赤道負荷は無視できると考えられています。この方法は、より大きな?...

The lens is a transparent and avascular organ in the anterior chamber of the eye that is responsible for fine focusing of light onto the retina. A clear basement membrane, called the lens capsule, surrounds a bulk of elongated fiber cells covered by an anterior monolayer of epithelial cells1,2. Life-long growth of the lens depends on the continuous proliferation and differentiation of epithelial cells at the lens equator into new fiber cells that are added onto previous generations of fiber cells in a concentric manner2. Over time, lens fiber cells undergo compaction, resulting in a rigid center in the middle of the lens called the nucleus1. Accommodation, defined as a dioptric change in the optical power of the eye, occurs in humans when the ciliary muscles contract to deform the lens and allow a true increase in optical power to focus on near objects3-5. In the unaccommodated eye, the lens is held in a relatively flattened state due to tension from zonular fibers. When the ciliary muscles contract, the tension on the lens is released, leading to decreased lens equatorial diameter and increased axial thickness. Age-related changes in the lens cause presbyopia, a progressive loss of lens accommodation, which leads to the need for reading glasses. 
Several studies have linked presbyopia to age-related increase in the intrinsic stiffness of the lens6-11. Stiffness is defined as the resistance of an elastic object to deform under applied load. A variety of methods have been used to examine stiffness of human lenses, including spin compression12-14, actuator compression15, probe indentation16, dynamic mechanical analysis 6,10 and bubble-based acoustic radiation force17. While mouse lenses do not accommodate or develop presbyopia, mouse models for lens pathologies are valuable tools because mice are less expensive than larger animals, well characterized genetically and undergo accelerated age-related changes due to rapid aging. A handful of studies have examined mouse lens stiffness with compression methods and demonstrated changes in lens stiffness due to aging or targeted genetic disruptions18-21. Thus, mouse lenses are good models for studying age-related changes in lens stiffness. 
This protocol describes a simple and inexpensive, yet precise and reproducible, compression method for determining mouse lens stiffness based on application of glass coverslips onto the lens in conjunction with photographing the lens through a dissection microscope and mirror. This method yields robust strain and morphometric data with an easily fabricated and assembled apparatus. The representative results confirm that mouse lenses increase in stiffness with age.

<table border="0" cellpadding="0" cellspacing="0" width="811">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Fine tip straight forceps</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">11252-40</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microdissection scissors, straight edge</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved forceps</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">11272-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seizing forceps</td>
			<td style="width:139px;">Hammacher</td>
			<td style="width:119px;">HSC 702-93</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">12565154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm petri dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">0875713A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X phosphate buffered saline (PBS)</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18mm x 18mm glass coverslips</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">12-542A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Measurement chamber with divots to hold lenses</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Custom-made (see Figure 1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Right-angle mirror</td>
			<td style="width:139px;">Edmund Optics</td>
			<td style="width:119px;">45-591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light source</td>
			<td style="width:139px;">Schott/Fostec</td>
			<td style="width:119px;">8375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Illuminated dissecting microscope</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:119px;">SZX-ILLD100</td>
			<td>With SZ-PT phototube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital camera</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">Coolpix 990</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequential application of glass coverslips to assess the compressive stiffness of the mouse lens: strain and morphometric analyses

眼のレンズは屈折し、網膜上に鮮明な画像を形成するために光を集光する透明な器官です。ヒトでは、毛様体筋の契約は、近くの物体に焦点を合わせるレンズの光学的パワー、宿泊として知られているプロセスの増加につながる、レンズを変形させます。加齢に関連するレンズ剛性の変化老眼にリンクされている、対応するレンズの能力の減少、および、拡張によって、老眼鏡が必要とされています。マウスのレンズは老眼を受け入れるか、発症しないにもかかわらず、マウスモデルが理解レンズ病理のための貴重な遺伝的なツールを提供することができ、およびマウスで観察された促進老化は、レンズ内の加齢変化の研究を可能にします。このプロトコルは、レンズに圧縮荷重を増加させる、順次適用し、ガラスカバースリップを使用して、マウスレンズ剛性を決定するために、簡単、正確、かつ費用効果的な方法を示します。代表的なデータは、マウスのレンズのような、年齢とともにより硬くなっていることを確認します人間のレンズ。この方法は、非常に再現性があり、潜在的により大きな動物から試験レンズを機械的にスケールアップすることができます。

剛性と2-、4-および8ヶ月齢のマウスのレンズの寸法を測定しました。マウスはTSRI動物飼育施設から得られた純粋なC57BL6株バックグラウンド上のすべての野生型動物であり、そして各レンズが1〜10のカバーガラスを装填しま​​した。軸方向および赤道株を各カバースリップを添加した後にレンズの軸と赤道直径を測定し、次いで対応するアンロード直径の直径の各変?...

Watch this Scientific Journal Video about Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses at JoVE.com

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

マウスレンズの圧縮剛性を評価するために、ガラスカバースリップの逐次適用：ひずみと形態計測分析

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

sequential-application-glass-coverslips-to-assess-compressive

Research

JoVE Journal

Biology

7.2K ビュー.  The Scripps Research Institute. Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

ビデオ: Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

レンズゼブラフィッシュでの移植とアイ変異体の解析への応用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

生物学

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

プロテオーム解析のためのマウスの硝子体と網膜の内臓摘出

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

相間細胞核の力学特性を探査する生物物理学的なアッセイ：基板の歪みのアプリケーションおよびマイクロニードル操作

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

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 ...

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.

形態とマウスの下顎頭軟骨細胞解析法

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

レンズ無料のビデオ顕微鏡、付着性細胞培養の定量的解析

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

電気生理学測定の電気中立運送者の活動を研究への応用

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...