プロジェクトは、国立心臓、肺、血液研究所NIH助成U01HL098180によってサポートされていました。内容はもっぱら著者の責任であり、必ずしも国立心臓、肺、血液研究所や国立衛生研究所の公式見解を表すものではありません

1。試薬のセットアップ 1.1解剖とイメージング媒体リーボビッツのL-15培地（L15）は、FBS（10％）およびペニシリン - ストレプトマイシン（100単位/ mlのペニシリンGナトリウムと硫酸ストレプトマイシン100μgの/ ml）を補足される気管試料の収穫と撮像時の両方で使用されている。 2。浅い城壁文化チャンバアセンブリ気管組織を保持するために使用される室は、 図1に示されている。室の床は35ミリ培養皿の底ガラスである。チャンバーの上面と側面をガラス皿の上に厚さ0.3mmのシリコーンシートの小片を配置することによって生成される。シートの縁部を丸底ディッシュに合わせてカットされ、中心がさらに（ステップ2.1〜2.3を参照）浅い中央チャンバを形成するようにトリミングされる。このアセンブリの上に18mmの円形のガラスカバースリップを撮像するのに適した密閉チャンバを生成するように配置されている。 <ol><li>完全に0.3ミリメートルの厚さのシリコーンシートの二乗は18ミリメートルラウンドガラスカバースリップをカバーしています。 </li><li>標準鋭い解剖ハサミでカバースリップ（シリコーンシートの円形の層になる）のエッジの周りから余分なシリコーンシートを切り落とす。 </li><li>鋭いメスを切り出し、覆われたカバースリップ（したがってイメージングチャンバーの上部と側面を形成する）の中間からシリコーンシート（約10mm角）の中央広場を取り除くと。 </li></ol> 3。気管ハーベストと準備<ol><li>ローカル機関動物のケアと使用委員会および/または政府のガイドラインに従ってマウスを安楽死させるとまで十分L15メディア水没組織（著者は妊娠の日16.5でちょうど出産前にマウスを使用している、へと標準的なプラスチック35ミリ培養皿に気管を削除、生まれたばかりの新生児、および成体動物2）。 </li><li>の外側に取り付け、非気管関連組織を除去鈍的切開を用いた気管。 </li><li>ただミクロ解剖ハサミ（ 図2A、1行目）を使用して、輪状軟骨下横カットで喉頭を削除します。 </li><li>ミクロ解剖はさみ（ 図2A、2行目）を使用して、ただカリーナ上記横カットで左右の主気管支の分岐を削除します。 </li><li>優しく細かい鉗子で気管を保持し、任意の粘液や血液をクリアするためにP1000とP1000ピペットマンチップ付き入浴L15培地〜1mlでルーメン2〜3回洗い流す。 </li><li>ミクロ解剖はさみ（ 図2B、1行目）と横方向のカットを使用した気管の3-4リングセグメントを切り取る。 </li><li>ミクロ解剖はさみ（ 図2B、2行目）を使用して、この短い気管セグメントの気管筋の中央を縦にカットします。 </li><li>ミクロ解剖はさみ（ 図2Cを使用して気管軟骨輪の中央を縦にカット</stroイメージングに適した気管組織の2つのセクション（つのセクションは、 図2（d）に示すように）残しNG&gt;、線）。 </li></ol> 4。繊毛イメージング<ol><li>転送気管組織セグメント、L-15培地100-200μLで35ミリのガラス底培養皿の真ん中の上にダウンルーメン側、。 </li><li>繊毛生成されたフローを定量化するには、気管の組織試料（〜1×10 11個/ ml以下Fluoresbrite 2.5％水溶液の微小球懸濁液を20μL/ ml）を入浴L-15培地に蛍光0.20μmの微小球の少量を追加します。 </li><li>シリコーンシートにカットウィンドウによって形成された壁に囲まれた浅い室の真ん中に位置気管サンプルと、イメージングチャンバーダウン気管サンプルシリコーンシート側の上部に（上記1〜3の手順を参照してください）先に調製を置きます（ 図1 ）。 </li><li>ゆっくりと所定の位置に固定するためにカバースリップを押し下げとエッジのボイジャーから余分なL-15メディアを取り出すGA P1000チップとピペットマンP1000。 </li><li> 100X客観的かつDIC光学系と倒立顕微鏡で35ミリのガラス底培養皿と気管サンプルをマウントします。 </li><li>気管筋の丸まっエッジ（：記録された映画の例では、静止画図2Dアウトラインボックス、E）の繊毛気管上皮に沿って毎秒+フレームは200で繊毛運動の映画を集め高速度カメラやムービー取得ソフトウェアを使用（ 補足ムービー1）。 </li><li> FITC落射蛍光イメージングと低照度CCDカメラ（上記の機器リストを参照）を使用して、繊毛生成フロー（ 補足作品2）の後の定量化を可能にするために気管繊毛上皮の表面を横切って蛍光ミクロス運動の映画を収集します。 </li></ol> 5。繊毛の運動の定量<ol><li> ImageJのソフトウェアパッケージにDIC画像化された映画をロードし、Iで説明する手法を以下AINドラモンド13鼓動繊毛を横断ラスタラインを描画するラインツールを使用します。このラインの &quot;再スライスは&quot;行に亘る繊毛運動が波形パターンを生成カイモグラフ型画像を生成する。分あたりのビート（ すなわちヘルツ）の数は、次に計算することができ、そこから、各波のピークの間のピクセル数、彼らが測定されます（1ピクセル= 1つのムービーフレーム）。 </li><li> ImageJのソフトウェアパッケージに繊毛、生成されたフロー負荷蛍光ビーズ映画を測定し、手動で気管上皮の表面を横切って蛍光ビーズを追跡し、ソフトウェアを追跡各ビーズのためのビーズの速度と方向性を生成させるMTrackJプラグ14を使用します。 </li></ol>破っ繊毛からの距離として流れを定量化するために強く測定流量振幅に影響を与えることができる蛍光ビーズを使用している場合は注意が必要です。補足ムービー2でビーズの動きはこの良い例です。この例では、ビーズ動きが鼓動繊毛の表面に高速で、かなり遠く入浴メディアでビーズのためにオフに低下します。これを制御するには、ビーズトレーシングは、唯一の正しい比較を行うことができるように、すべてのサンプルで繊毛上皮上の固定された距離で収集されるべきである。最終的に、流体の流れに代表的なデータを得るために、ビーズをできるだけ長く追跡されるべきであり、我々は、流体の流れを算出するビード10を覆うトラック+繊毛細胞を使用することを好む。

<ol>
	<li>Stannard, W., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+function+and+the+role+of+cilia+in+clearance.">Ciliary function and the role of cilia in clearance.</a> J. Aerosol. Med. 19, 110-115 (2006).</li><li>Francis, R. J., 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=The+initiation+and+maturation+of+cilia+generated+flow+in+the+newborn+and+postnatal+mouse+airway.">The initiation and maturation of cilia generated flow in the newborn and postnatal mouse airway.</a> American Journal of Physiology: Lung Cellular and Molecular Physiology. 296, 1067-1075 (2009).</li><li>Aune, C. N., 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=Mouse+model+of+heterotaxy+with+single+ventricle+spectrum+of+cardiac+anomalies.">Mouse model of heterotaxy with single ventricle spectrum of cardiac anomalies.</a> Pediatric Research. 63, 9-14 (2008).</li><li>Tan, S. Y., 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=Heterotaxy+and+complex+structural+heart+defects+in+a+mutant+mouse+model+of+primary+ciliary+dyskinesia.">Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia.</a> J. Clin. Invest. 117, 3742-3752 (2007).</li><li>Zhang, Z., 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=Massively+parallel+sequencing+identifies+the+gene+Megf8+with+ENU-induced+mutation+causing+heterotaxy.">Massively parallel sequencing identifies the gene Megf8 with ENU-induced mutation causing heterotaxy.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 3219-3224 (2009).</li><li>Caruso, G., Gelardi, M., Passali, G. C., de Santi, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+scraping+in+diagnosing+ciliary+dyskinesia.">Nasal scraping in diagnosing ciliary dyskinesia.</a> Am. J. Rhinol. 21, 702-705 (2007).</li><li>Leigh, M. W., Zariwala, M. A., Knowles, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ciliary+dyskinesia:+improving+the+diagnostic+approach.">Primary ciliary dyskinesia: improving the diagnostic approach.</a> Curr. Opin. Pediatr. 21, 320-325 (2009).</li><li>Delmotte, P., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+beat+frequency+is+maintained+at+a+maximal+rate+in+the+small+airways+of+mouse+lung+slices.">Ciliary beat frequency is maintained at a maximal rate in the small airways of mouse lung slices.</a> Am. J. Respir. Cell Mol. Biol. 35, 110-117 (2006).</li><li>Choe, M. M., Tomei, A. A., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+3D+tissue+model+of+the+airway+wall+and+mucosa.">Physiological 3D tissue model of the airway wall and mucosa.</a> Nat. Protoc. 1, 357-362 (2006).</li><li>Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R., Randell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-differentiated+human+airway+epithelial+cell+cultures.">Well-differentiated human airway epithelial cell cultures.</a> Methods Mol. Med. 107, 183-206 (2005).</li><li>You, Y., Richer, E. J., Huang, T., Brody, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+mouse+tracheal+epithelial+cells:+selection+of+a+proliferative+population.">Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 283, 1315-1321 (2002).</li><li>Thomas, B., Rutman, A., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupted+ciliated+epithelium+shows+slower+ciliary+beat+frequency+and+increased+dyskinesia.">Disrupted ciliated epithelium shows slower ciliary beat frequency and increased dyskinesia.</a> Eur. Respir. J. 34, 401-404 (2009).</li><li>Drummond, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+cilia+in+zebrafish.">Studying cilia in zebrafish.</a> Methods Cell Biol. 93 (08), 197-217 (2009).</li><li>Meijering, E., Dzyubachyk, O., Smal, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+cell+and+particle+tracking.">Methods for cell and particle tracking.</a> Methods Enzymol. 504, 183-200 (2012).</li><li>Sisson, J. H., Stoner, J. A., Ammons, B. A., Wyatt, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-digital+image+capture+and+whole-field+analysis+of+ciliary+beat+frequency.">All-digital image capture and whole-field analysis of ciliary beat frequency.</a> J. Microsc. 211, 103-111 (2003).</li><li>Schwabe, G. C., 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=Primary+ciliary+dyskinesia+associated+with+normal+axoneme+ultrastructure+is+caused+by+DNAH11+mutations.">Primary ciliary dyskinesia associated with normal axoneme ultrastructure is caused by DNAH11 mutations.</a> Hum. Mutat. 29, 289-298 (2008).</li><li>Shah, A. S., 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=Loss+of+Bardet-Biedl+syndrome+proteins+alters+the+morphology+and+function+of+motile+cilia+in+airway+epithelia.">Loss of Bardet-Biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 3380-3385 (2008).</li><li>Clary-Meinesz, C. F., Cosson, J., Huitorel, P., Blaive, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effect+on+the+ciliary+beat+frequency+of+human+nasal+and+tracheal+ciliated+cells.">Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells.</a> Biology of the Cell / under the auspices of the European Cell Biology Organization. 76, 335-338 (1992).</li></ol>

著者らは、開示することは何もありません。

繊毛のビート周波数（CBF）の測定は、高電力顕微鏡対物レンズと高速画像集録ハードウェア13,15を使用して比較的容易であり、かつCBF測定は健康と病気の間に粘液線毛クリアランスを調査するほとんどの研究の基礎を形成する理由を説明します。しかし、測定するのがより困難であり、どちらも毛様体、生成されたフローと繊毛ビート波形の両方の基礎と重要性を、無視CBF測定だけで...

気道上皮における運動性繊毛機能の分析は、粘液線毛クリアランスと肺の健康1に影響を与えることができる遺伝的要因と環境要因を解明するための実験が重要である。イメージングのために開発されたシンプルなプロトコルマウス気道上皮は、変異とノックアウトマウスモデルで気道繊毛運動を尋問するために効率的な方法を提供し、マウス気管組織切開と高解像度ビデオ顕微鏡による気道繊毛運動のex vivoでのイメージングだけで基本的なスキルを必要とする。このプロトコルはheterotaxy 2-5関連付けられた先天性心疾患を持つ変異体では運動性繊毛機能の迅速な評価（繊毛ビート周波数、繊毛ビート形状、繊毛生成されたフロー）を可能にするために大規模マウス突然変異誘発画面中に確立され、洗練された。 気道繊毛運動性を研究するために使用される現在の技術は、急性エクスビボ型またはイオンのいずれかに分類することができ試験管内実験的アプローチでゲル用語。急性実験は人間の鼻/気道ブラシ生検6,7とシンプルな横気道セクション8の分析のex vivoでの可視化が含まれています。 インビトロのアプローチは、空気液界面培養物または気道の懸濁培養物9-11と同様に分化繊毛上皮のシートを生成するために、様々な細胞培養技術を利用する。任意の使用可能な繊毛上皮細胞が実験（4-6週9,10）のために制作される前に、しかし、これらの気道上皮reciliation技術は時間と訓練に非常に多額の投資を必要とする。気道上皮ブラシ生検の急性ex vivoでの分析は、一般的にヒト臨床試験に使用されているが、この方法は、機械的な悪化組織傷害12によるマウスの研究において使用できない。 覚書の分析のために、このプロトコルで概説技術電子気管気道上皮は、実行するだけで簡単ではありませんが、それは特別な解剖のスキルも、ビデオ顕微鏡による撮影のためにそれらの標準以外の任意の特殊な機器を必要としません。この単純なプロトコルには多くの利点がある。マウス気管組織の収穫は、実行するために迅速かつ簡単であるように、まず、それはマウスの大規模な数の気道繊毛機能の迅速な評価が可能になります。これは、in vitroでの治療で別の短期的な影響の急性分析を含めることができます。第二に、ex vivoでの技術であること、繊毛気道上皮は、それがサポートしている組織を根底に接続されたまま、したがって、関連する細胞シグナル伝達経路を保持します。従って、 インビトロ reciliated気道上皮内と比較して、この製剤は、 生体組織環境における自然のより良い表現である。第三に、このプロトコルは繊毛運動fの客観的な評価を与えることができる種々の定量的なパラメータの数の取得を可能にする慰め。最終的に、気道繊毛の可視化のための他の現行の方法とは対照的に、このプロトコルは、繊毛ビートと異時波発生の高分解能イメージングのための最適な繊毛のプロファイルビューを可能にする、繊毛ビート方向に対して直角繊毛の可視化を可能にする。 このプロトコルは、例えば、薬剤、遺伝的要因、環境曝露、および/または気道繊毛機能と生成/維持にかかる粘液負荷等の機械的要因の役割などの実験、幅広いニーズに対処するための多くの方法で変更することができる気道繊毛ビートと異時波動伝播。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 Medium </td>
 <td>Invitrogen </td>
 <td>21083-027 </td>
 <td>No phenol red </td>
 </tr>
 <tr>
 <td>Fetal bovine serum </td>
 <td>Hyclone </td>
 <td>SH30088.03 </td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin </td>
 <td>Invitrogen </td>
 <td>15140-122 </td>
 <td></td>
 </tr>
 <tr>
 <td>2x fine forceps </td>
 <td>Roboz </td>
 <td>RS-4976 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5676 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5620 </td>
 <td></td>
 </tr>
 <tr>
 <td>Scalpel </td>
 <td>Roboz </td>
 <td>RS-9801-15 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 pipetman </td>
 <td>Gilson, Inc </td>
 <td>F123602 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 tips </td>
 <td>Molecular BioProducts </td>
 <td>2079E </td>
 <td></td>
 </tr>
 <tr>
 <td>18 mm round glass cover slips </td>
 <td>Fisher Scientific </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic 35 mm culture dishes </td>
 <td>Corning </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Glass bottom 35 mm culture dishes </td>
 <td>Warner Instruments </td>
 <td>W3 64-0758 </td>
 <td></td>
 </tr>
 <tr>
 <td>Silicone sheet 0.012" (0.3 mm) thick </td>
 <td>AAA Acme Rubber Co </td>
 <td>CASS-.012X36-63908 </td>
 <td></td>
 </tr>
 <tr>
 <td>0.20 &mu;m diameter Fluoresbrite YG Carboxylate Microspheres </td>
 <td>Polysciences </td>
 <td>09834-10 </td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope, with 100x oil objective and DIC filters </td>
 <td>Lecia</td>
 <td>DMIRE2 </td>
 <td>Brand is not critical. </td>
 </tr>
 <tr>
 <td>100-watt mercury lamp, epifluorescent FITC excitation/emission filters</td>
 <td>Lecia </td>
 <td></td>
 <td>Brand is not critical.</td>
 </tr>
 <tr>
 <td>Microscope stage Incubator</td>
 <td>Lecia </td>
 <td>11521749 </td>
 <td>Not required if imaging cilia at room temperature</td>
 </tr>
 <tr>
 <td>High-speed camera bright field </td>
 <td>Vision Research </td>
 <td>Phantom v4.2 </td>
 <td>Brand is not critical. Must be faster than 125 fps</td>
 </tr>
 <tr>
 <td>High-speed fluorescent camera </td>
 <td>Hamamatsu </td>
 <td>C9100-12 </td>
 <td>Brand is not critical. Must be faster than 10 fps</td>
 </tr>
 <tr>
 <td>Movie analysis software</td>
 <td>National Institutes of Health</td>
 <td>ImageJ with MtrackJ plugin</td>
 <td></td>
 </tr>
 </tbody>
</table>

ex vivo method for high resolution imaging of cilia motility in rodent airway epithelia

粘膜毛様体クリアランス機能にとって重要な洞察繊毛運動機能を定量的に分析するための撮像マウス気道上皮ためのex vivoでの技術が確立されている。収穫されたばかりのマウス気管気管の筋肉を長手方向にカットしたガラス底皿に浅い薄肉チャンバー内に装着されている。気管サンプルは長手方向にカールする気管筋を活用するために、その長軸に沿って配置されている。これは全体の気管の長さに沿ってプロファイルビューで繊毛運動のイメージングを可能にします。 200フレーム/秒のビデオは繊毛ビート周波数と波形毛様体の定量分析を可能にするために微分干渉顕微鏡と高速デジタルカメラを用いて得られる。撮影時の蛍光ビーズを添加しても、流体の流れを生成繊毛を決定することができる。プロトコル時間は室の準備のために5分で約30分、サンプルの5〜10分間に及ぶ取り付け、およびビデオ顕微鏡のための10-15分。

コントロール気道繊毛がはっきり見えるようにして調整された方法（ 補足ムービー1、ムービーの再生が15％をリアルタイムに減速されています）でビートに見えるはず繊毛ビート（ 補足作品2の方向に顕著な流れに、、ムービーの再生は100％ですリアルタイム）。繊毛映画の定量は、 図3に見られるものと同様の結果が得られるはず。蛍光ビーズの追跡?...

Watch this Scientific Journal Video about Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia at JoVE.com

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

気道繊毛運動と繊毛生成されたフローをマウス気管を使用して可視化し、定量化するための簡単​​で信頼性の高い技術が記載されている。この技術は様々な要因が薬理学的薬剤、遺伝的要因、環境曝露、及び/又は粘液負荷などの機械的要因を含む、繊毛運動性にどのように影響するかを決定するために変更することができる。

齧歯気道上皮における繊毛の運動性の高分解能イメージングのためのex vivo法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

ex-vivo-method-for-high-resolution-imaging-cilia-motility-rodent

Research

JoVE Journal

Biology

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

20.7K ビュー.  University of Pittsburgh. 気道繊毛運動と繊毛生成されたフローをマウス気管を使用して可視化し、定量化するための簡単​​で信頼性の高い技術が記載されている。この技術は様々な要因が薬理学的薬剤、遺伝的要因、環境曝露、及び/又は粘液負荷などの機械的要因を含む、繊毛運動性にどのように影響するかを決定するために変更することができる。

ビデオ: Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

成長円錐の高分解能イメージングのためのアメフラシから神経細胞の培養

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

アダルトキイロショウジョウバエのロコモーションの高解像度のビデオトラッキング

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

小説<em&gt;エクスビボ</emマウス胎児ハート&gt;文化法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

運動の時空間マッピングで<em&gt;エクスビボ</em腸の&gt;準備

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

C-FOSタンパク質免疫組織学的検出：特定の生理反応に関与する中央経路のマーカーとして有用ツール<em&gt;インビボ</em&gt;と<em&gt; ex vivoで</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

IRF3のリン酸化依存性活性化を監視するために、高解像度の方法

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

出芽酵母における高解像度画像と個人アストラル微小管動態の解析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...