この作品は、私たちは、SLO発現プラスミドのためのオクラホマ大学から博士R. Twete​​nに感謝NWAにNIHの助成金R37 AI34867およびR01 GM064625によってサポートされていました。

1。 ASMの転写サイレンシング<ol><li>シード1.5×10 35mmのガラスボトムディッシュ（MatTek社製）上のDMEM増殖培地（10％ウシ胎児血清（FBS）を有するDMEM高グルコース、2mMのL-グルタミン、1％ペニシリン-ストレプトマイシン）2ml中の5 HeLa細胞および5％CO 2インキュベーター内で37℃で一晩インキュベートする。 </li><li>翌日、増殖培地をオフ吸引し、siRNAトランスフェクション混合物を添加する前に2ミリリットルのDMEMを減少血清培地（4％FBSを含むDMEM）、少なくとも1時間に交換してください。 </li><li> siRNAのオリゴトランスフェクション混合物のための4のチューブを準備します。チューブAとCに、トランスフェクトされる35mmディッシュあたり、血清および4μlのトランスフェRNAiMAXトランスを減少させた250μLのOptimemを追加。チューブBは、オプティメム250μlのを追加し、血清およびトランスフェクトされる35ミリメートル皿あたり制御メディアGC含量のオリゴ（対照siRNA）を8μlの（160ピコモル）、減少した。チューブDでは、追加のOptimem250μlの35ミリメートル皿Tあたり、SMPD1オリゴ（siASM）の血清および8μL（160ピコモル）を削減Oトランスフェクトする。室温で5分間チューブをインキュベートする。 </li><li>チューブAおよびBは、トランスフェクションのためのコントロールのsiRNA複合体、およびASM siRNAのための管CおよびDを形成する。 20分間室温で反応をインキュベートする。 </li><li>それぞれ35 mmのガラスボトムディッシュにコントロールsiRNAとASMのsiRNAトランスフェクション反応にゆっくりと混合物は、滴下して、追加し、37℃/ 5％CO 2でインキュベートする。 24時間のトランスフェクション後に、静かに、メディアをオフに吸引し、新鮮なDMEM増殖培地と交換してください。効率的なASMのノックダウンのために、料理はさらにライブイメージング前に約31時間（合計55時間）、37℃/ 5％CO 2でインキュベートする必要があります。 </li></ol> 2。ライブ顕微鏡用試薬の調製<ol><li> Ca 2 +およびMg 2 +なしで冷たい1×PBS中100 NG /μLの濃度で組換え孔形成毒素ストレプトリジンO（SLO）の溶液を調製する。これらのアッセイにおいて使用されるSLOはconstitutivelある親切に博士ロッドTwete​​n、オクラホマ大学が提供する活動のための還元剤は必要ありませんYアクティブシステイン変異体。毒素は、E.で発現された大腸菌 BL21 DE3、以前に16記載されているように、ネイティブな条件下で精製。 </li><li> 25 mM溶液を作成するために、DMSOに再懸濁FM1-43凍結乾燥粉末：FM1-43のストック溶液を調製する。 </li><li>溶液Aを準備します。4μmの最終濃度にCa 2 +を冷DMEM中でFM1-43のストック溶液を希釈（1.5ミリリットルをマテック皿のあたりに必要とされている）と、必要になるまで氷上で保存する。 </li><li> B液を準備します。4μM（マテックディッシュ当たりに必要な1.5ミリリットル）の最終濃度になるようにCa 2 +および10のEGTA（Ca 2 +を含まないDMEM）なしで冷たいDMEM中でFM1-43ストック溶液を希釈し、まで氷上に保存する必要としていました。 </li><li>事前暖かい（37℃）エッペンドルフチュー​​ブ内の溶液Aと溶液Bを1ミリリットルずつ。 </li><li>のCa 2 +、およびCを含むDMEMのアイスソリューションを保つ2 +を含まないDMEM中。 </li></ol> 3。 FM1-43の処理されたSLOに流入し、未処理細胞の生細胞イメージング<ol><li> 、砕いた氷でMサイズの浅い金属製の容器を埋める氷を持つ大規模なガラス容器に、それを反転させ、露出した金属容器の底面のみを残して、追加の氷を加える。でも、熱転写を可能にするために露出した金属底に湿ったペーパータオルを置きます。 </li><li> 1コントロールsiRNA処理マテック皿（ サンプル1）を取り、冷たい湿ったペーパータオルの上に置きます。優しくすべてのメディアをオフに吸引し、寒さのCa 2 +を含まないDMEMで3回洗う。最後の洗浄の後、皿にすべてのメディアをオフに吸引除去する。 </li><li>のCa 2 +の存在無しSLO創傷（ サンプル1）と細胞内の画像細胞関連FM1-43に、35 mmのガラスボトムディッシュの中心ガラスカバースリップに冷溶液Bの180μLを加え、5インキュベート氷上分（ 表1）。 </li&gt; <li> 37℃に加熱し、（温度、湿度、CO 2レベルを維持する）環境室を搭載した共焦点顕微鏡のステージ上マテック皿を置きます。 40X、NA 1.3の対物レンズ（ニコン）を通して見て画像化される細胞のフィールドを検索します。可能な場合、熱ドリフトを補正するためにPerfectFocusまたは類似のデバイスをオンにします。 </li><li>そっと35-mmディッシュの側に予熱溶液Aの1ミリリットルを加え、環境室（ 表1）のカバーを交換してください。 </li><li> （ULTRAVIEW近所パーキンエルマースピニングディスク共焦点顕微鏡システムにおいてVolocity）適切なイメージングソフトウェアを使用して、すべての3秒1コマで4分間の画像を取得する。 FM1-43の励起は488nmのバンドパスダイクロイックフィルタとを用いて488nmのレーザー線を用いて達成され、発光判別は527（W55）発光フィルターを使用することによって達成される。レーザパワーレベルとカメラの感度は、100ミリ秒の画像露光を達成するように設定されている。 </li><li>繰り返します3.2〜3.6は、そのステップ3.5溶液Bである（ 表1）を追加する必要がありますを除いて、Ca 2 +の非存在下ではありませんSLO創傷（ サンプル2）を対照siRNA処理細胞でFM1-43を検出するには、手順。 </li><li>プレゼンス（ サンプル3）またはCa 2 +が存在しない場合（ サンプル4）の中心ガラスカバースリップにSLOを含有する冷溶液Bの180μlを添加するのいずれかでSLOを負傷したコントロールsiRNAで処理した細胞へのFM1-43の流入を測定するには35 mmのガラスボトムディッシュ、ステップ3.3のように氷上で5分間インキュベートする。繰り返します3.6を通じて3.2ステップ、ステップ3.5（ 表1）で溶液AまたはB液のいずれかを追加し調整します。 </li><li>原形質膜修復におけるASMの役割を決定するには、手順は、すべての条件（ 試料5-8）（表1）のためのASM siRNAで処理した細胞を使用して3.2から3.6を繰り返します。 </li><li>上の細胞外組換えスフィンゴミエリナーゼ（SM）の役割を決定するために、（FM1-43の流入における阻害によって反射された）、細胞膜修復の動態は、（ サンプル9月10日 ）、SMが追加されたステップ3.5（0〜50μU）に溶液Aのどちらか、または溶液Bとし、生細胞の間に細胞に添加されている1ミリリットル（ 表1）の総体積で撮像。 </li></ol> 4。細胞へのFM1-43流入の定量化<ol><li>ムービーが取得された後、画像解析ソフト（Volocityスイート、PerkinElmer）を用いFM1-43の細胞内蛍光強度を測定する。フィールド内の各細胞の細胞質ゾル領域に関心領域を描画し、動画の全フレームを通じて平均FM1-43の蛍光強度を測定するためにソフトウェアツールを適用する。 </li><li>初期FM1-43染色の変動を調整するには、各ビデオのためのデータは、経時的な蛍光強度の増加倍数として表現さ​​れ、それぞれの異なる条件に対してプロットされている。個々の時点あたりの蛍光の倍増加セルは、F Tは、特定の時点での平均蛍光であり、F 0が t = 0（ 図1-2）での平均蛍光であるF T / F 0として計算される。 </li></ol>

<ol>
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著者は、彼らが競合する金融利害関係はありません宣言します。

機械的損傷と、細胞膜の修復に関する初期の研究では、ガラスビーズ、マイクロピペット、せん断、傷や削れを落とすに至るまで、負傷した細胞の多様なメカニズムに依存していました。すべてのこれらのアッセイの読み出しは、定性的ではなく定量的であり、修復機構の動力学に関する正確な情報を与えなかった。損傷のこれらの形態の後に原形質膜を再シールする細胞の能力は、細胞質?...

これは、機械的損傷後の血漿膜修復は、Ca 2 +依存性プロセスであること1,2数十年前から知られている。その後の研究は、傷口を通してのCa 2 +流入は、3,4を再密封するために必要とされる傷害の部位で細胞内小胞のエキソサイトーシスの積極的なプロセスをトリガすることが示された。細胞の細胞質内にロード蛍光色素の損失の速度は、修復の速度を評価するために使用され、再封が損傷後5 &lt;30秒以内に完了したと結論された。二つのモデルは、最初は、細胞膜修復にエキソサイトーシスのための要件を説明するために提案された。病変を通じてのCa 2 +流入は、そのだろう大規模な「パッチ」を形成、細胞内小胞の最初に同型の融合を引き起こすことが示唆さ1）「パッチ」モデル、次いで、創傷6と、その膜を提案しADDE 2）張力低減モデルを再シールするために原形質膜に適用すること創傷の近傍におけるCa 2 +依存性エキソサイトーシスによって7日間二重層の再封を容易にする、原形質膜の張力を減少させるであろう。原形質膜修復におけるCa 2 + -誘発さエキソサイトーシスの役割は、リソソームが損傷菌におけるそれらのエキソサイトーシスおよび原形質膜の修復を促進するのCa 2 +センサー分子、シナプトタグミンVIIを含有することを示す研究によって補強された8,9,10,11 。 しかしながら、追加の証拠は、単独で、エキソサイトーシスは、形質膜の修復を促進するのに十分ではなかったことを示した。原形質膜上の機械的な涙液に加えて、細胞損傷の頻繁な形態は、細菌12,13又は14,15免疫細胞によって産生さ孔形成毒素による透過性である。機械的な涙とは違って、孔形成タンパク質は、細胞膜は、膜「パッチ」を適用するか、削減するだけで再密封することはできない、安定した、タンパク質が並ぶ細孔を形成する上で自分自身を挿入膜張力をING。興味深いことに、研究は、哺乳類細胞は孔形成タンパク質を用いて透過処理した後に、それらの原形質膜を修復するための効率的な機構を有することを明らかにし、このプロセスは、細胞外Ca 2 + 12の存在を必要とする。機械的な傷5で観察されたので、この発見は、細胞表面から貫通孔の除去にも迅速なプロセスであったかどうかという疑問を提起した。プロセスは、細胞外Ca 2 +を必要とし、約30秒以内に完了する：驚くべきことに、我々の最近の研究では、孔形成毒素で透過処理した細胞の再シールは、機械的創傷の修復に非常に類似した性質を有することが明らかになった。より詳細には、このプロセスを調査し、我々は最近、リソソームのCa 2 +調節性エキソサイトーシスに加えて、原形質膜の修復は、リソソーム酵素酸性スフィンゴミエリナーゼ（ASM）の放出によって引き起こさ必須であるエンドサイトーシスの急速な形態を含むことを学ん目のためだけでなく、貫 ​​通孔のEの除去だけでなく、機械的な傷16の補修用。 孔形成タンパク質との透過処理後の細胞再密封の動態を決定するために、我々の研究室では、レーザー損傷孤立筋繊維17の再シール性を評価するために以前に使用されていたライブイメージングの方法論を適応。このアッセイは、安定して蛍光強度が増加した脂質二重層の外側のリーフレット中にインターカレートする親油性色素FM1-43の特性に依存する。ときに、原形質膜二重層を形質膜損傷および修復18,16,19,20を検出するための感度の高いアッセイを提供し、細胞内膜への細胞外の色素アクセスして破壊される。孔形成タンパク質との透過処理後の細胞の再シール性の評価のためにこのアッセイを適合させるために、我々は、膜コレステロール21に結合する細菌毒素ストレプトリジンO（SLO）、4℃で細胞をプレインキュベートした。同期セル透過化は、その後容易に貫通孔の形成をもたらすコンフォメーションの変化をオリゴマー化および活性化する加熱された顕微鏡ステージ、媒体を温めるために氷から細胞を移動させることによって達成することができる。このアプローチの利点は、レーザー創傷を用いて以前に公開されたアッセイの上に、多数の細胞は、細胞集団のより良好なサンプリングを提供し、顕微鏡視野内で同時に分析することができることである。孔形成毒素との機械的損傷および透過後に見られる細胞の再シール工程の間の機構的類似点を考えると、我々はここで説明するアッセイは、細胞膜修復の基本的なプロセスに関与する因子を解剖するために、非常に汎用性と強力な方法を提供する。一例として、我々はそれが修復過程ののCa 2 +依存性および非依存性の手順を識別するために、このアッセイを使用することが可能であることを示している。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>HeLa 229 cell line</td>
			<td>ATCC</td>
			<td>CCL2-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose </td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose No Calcium</td>
			<td>Invitrogen</td>
			<td>20168</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Optimem Reduced Serum</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Control medium GC content RNAi oligo</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SMPD1 RNAi oligo</td>
			<td>Invitrogen</td>
			<td>HSS143988</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum Heat-inactivated</td>
			<td>Gemini-Bioproducts</td>
			<td>100-106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sphingomyelinase from Bacillus cereus</td>
			<td>Sigma</td>
			<td>S7651</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm-glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-14</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C9100-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spinning disk confocal microscope</td>
			<td>PerkinElmer</td>
			<td>UltraViewVoX</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software analysis software</td>
			<td>PerkinElmer</td>
			<td>Volocity Suite</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Environmental chamber</td>
			<td>Pathology Devices</td>
			<td>LiveCell System Chamber</td>
		 </tr>
		</tbody>
 </table>

live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds

原形質膜の損傷が頻繁なイベントであり、創傷は急速に細胞の生存を確実にするために修復されなければならない。のCa 2 +の流入は、30秒以内〜原形質膜上に機械的な創傷の修復をトリガする重要なシグナル伝達事象である。最近の研究では、哺乳動物細胞はまた、細孔が細孔エンドサイトーシスに続いてリソソーム酵素酸性スフィンゴミエリナーゼのエキソサイトーシスを伴うのCa 2 +依存性プロセスでの毒素の形成に透過処理後に原形質膜を再シールすることを明らかにした。ここでは、毒素ストレプトリジンOにより透過性細胞の再シールは、Ca 2 +流入にも迅速かつ依存していることを示すために使用される方法について説明します。アッセイデザインは、損傷事象および撮像により原形質膜の完全性を復元し、親油性色素FM1-43は細胞内膜に到達することによって程度を定量化する細胞の能力の正確な速度論的測定の同期を可能にする。このライブアッセイALSoは外因的能力の評価は、それらの感受性形質膜を修復する細胞の能力を反映して、FM1-43の流入を抑制するために、例えばスフィンゴミエリナーゼのような可溶性因子を添加することができる。このアッセイは、私たちは、酵素の細胞外のほかは、Ca 2 +が存在しない場合に透過性細胞の再シールを促進するため、スフィンゴミエリナーゼは、カルシウム2 +依存性エキソサイトーシスの下流で作用することを初めて示すことができました。

リソソーム酸性スフィンゴミエリナーゼで枯渇細胞における細菌スフィンゴミエリナーゼ（SM）レスキュー形質膜修復の低濃度。 FM1-43画像化アッセイを用いて、我々は、以前のCa 2 +の存在下での創傷のリソソーム酵素ASM欠損とSLOに曝露された細胞は、原形質膜の欠陥は、組換えヒト酵素19の細胞外添加によりレスキューされていることを示した。ヒトリソ?...

Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

親油性色素FM1-43に曝露された細胞のライブイメージングは​​、孔形成毒素は原形質膜から除去されることにより、反応速度の正確な決定を可能にする。これは、Ca 2 +、スフィンゴミエリナーゼおよび原形質膜の修復に関する他の要因のための要件を評価するために使用できる高感度のアッセイである。

カルシウムの役割を評価するためのイメージングアッセイを生きる<sup&gt; 2 +</sup孔形成毒素の傷の修復&gt;とスフィンゴミエリナーゼ

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

live-imaging-assay-for-assessing-roles-ca2-sphingomyelinase-repair

Research

JoVE Journal

Biology

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

11.6K ビュー.  University of Maryland. 親油性色素FM1-43に曝露された細胞のライブイメージングは​​、孔形成毒素は原形質膜から除去されることにより、反応速度の正確な決定を可能にする。これは、Ca 2 +、スフィンゴミエリナーゼおよび原形質膜の修復に関する他の要因のための要件を評価するために使用できる高感度のアッセイである。 

ビデオ: Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

の調製ショウジョウバエ</emハンギングドロッププロトコルを使用してライブイメージングのための>胚

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

イメージングカルシウムのためのテクニック 2 +人間の精子でシグナリング

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

電子スピン共鳴酸素マッピングのためのライブ種のマイクロイメージング

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

siRNAを介した遺伝子サイレンシングと組み合わせることで生細胞のカルシウムイメージングでは、CAを識別 2 + ER膜のリークチャネルとその調節機序

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

頭蓋間葉の細胞挙動を評価するための外植片アッセイ

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

初期のオートファジーイベントの細胞イメージングライブOmegasomesを以降

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

モザイク大人のライブイメージングのためのトマト/ GFP-FLP / FRT方法<em&gt;ショウジョウバエ</em&gt;視細胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

裏打ちクリア: 組織の脂肪組織の三次元イメージング法をクリア

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...