私たちは、ヘルプと、これらのプロトコルを設定するために助言を博士カール·ハインツ·クラウスとヴィンセントジャケに感謝していて、また、それらの技術的なサポートのためにNCCR博士ナビンGopaldassのバイオイメージングプラットフォームからクリストフ·バウアーとジェローム若いアカシカの未発達の枝角に感謝します。研究は、スイス国立科学財団のProDoc助成金によってサポートされています。

1。可視化とファゴソーム内のROS生産の定性的測定 OBGコーティングしたビーズは、事前に準備する必要があります。ビーズコーティング手順および寒天オーバーレイ技術が公表された参考文献5,14から適応されます。 <ol><li> 1.5ミリリットルチューブに3.0ミクロンのカルボキシル化シリカビーズ（約1.8×10 9ビーズ）の1ミリリットル懸濁液を添加し、ビーズを迅速にスピンし、ボルテックスにより1mlのPBSで3回洗う。 </li><li> 25 mg / mlのシアナミドを含有する1mlのPBSでビーズを再懸濁し（共有結合前標識BSAに結合することがカルボキシル化シリカビーズを活性化するため）、15分間ホイール上でインキュベートする。 </li><li>ウォッシュクイックスピンカップリング緩衝液1ミリリットル（0.1Mホウ酸ナトリウム、pH8.0）で3倍（あるいはテーブルのフルスピードで遠心5秒トップのミニ遠心機や、テーブルトップ遠心機で1分間2000×gで遠心分離）し、過剰シアナミドを削除するためにボルテックス。 </li><li> C500μlで洗浄したビーズをミックスOxyBurstグリーン1mgの（OBG）H2HFF（ジヒドロ-2 &#39;、4,5,6,7,7&#39; - hexafluorofluorescein）-BSAを含むバッファをoupling、キャッピング前に、標準ガス缶から窒素ガスでチューブを記入し、暗闇の中で、室温で14時間（一晩）ホイール上に静置します。 </li><li>ビーズは（PBS中にグリシン250 mm）が素早く回転し、ボルテックスによりクエンチング緩衝液を除去するために、カップリング緩衝液で2回、未反応のOBGを削除し、バッファを消光1ミリリットルで2回洗う。 </li><li> BSAに結合させるためにアレクサフルオロ594スクシンイミジルエステル50μgのを含むカップリング緩衝液1ミリリットルを加える。キャッピング前に窒素でチューブを記入し、暗所で室温で1.5時間、ホイール上に静置します。 </li><li>ビーズが反応を停止させ、迅速にスピンし、ボルテックスによりバッファを消光1ミリリットルで3回洗浄し、ビーズは、迅速にスピンし、ボルテックスにより1mlのPBSで3回洗浄します。 </li><li>最後に、長期保存のためのVアジド/ wの10％の2μlのPBS 1mlにビーズを再懸濁する。 concentratioを測定血球計数器（通常は約1〜2×10 9ビーズ/ ml）で懸濁液中のビーズのnは、キャッピング前に窒素でチューブを記入し、暗闇の中で、4℃で保存する。 </li><li> OBGフルオレセインの塗着効率は、500nmでの励起を用いて発光スペクトルをチェックすることによって試験することができる。西洋ワサビペルオキシダーゼ（HRP）の存在下でH 2 O 2で酸化すると、酸化されたビーズの蛍光強度は、発光ピーク（538nmで）で、非酸化コーティングされたビーズのそれよりも11〜12倍高い。 </li><li>収穫は指数関数的に光学的に透明なプラスチックやガラス底で3センチディッシュ上の細胞の密度の異なるプレートと、一晩、それらを成長さを10cmシャーレから細胞性粘菌細胞を増殖させる。実験の約80％コンフルエントである料理を選択してください。 細胞性粘菌の細胞を培養するための詳細なプロトコルは、15を公表されている。 </li><li> LoFlo媒体（LF培地）で、培地を交換し、インキュベートHL5C培地の細胞外およびエンドソーム内の自家蛍光を低減するために、実験の前に2時間。 </li><li>並行して、10〜15待つ、約1mmの厚さの寒天層を形成するために、平坦面（×10cmの10cmの、すなわちガラス板）上に寒天を入れ、LF培地10mlに1.5％バクト寒天を溶かす分固化させる。後で使用するためのLF媒体が2cm×2センチメートルの正方形、所定の位置に寒天層をカット。 </li><li>一方、回転ディスクまたは広視野顕微鏡を用意し、22℃の環境室の温度を設定し、この実験に必要な設定を行います（議論の中で、追加のコメントを参照してください）​​。 </li><li>インキュベーションの2時間後に3 cmディッシュからLF培地を吸引するが、細胞単層は依然として媒体の薄膜によって覆われるべきである。 1.5×10 7ビーズ/ mlにコーティングされたビーズを希釈し、細胞層の上に10μlを加える。 </li><li> 1平方寒天シートを取り、余分な液体を排出ウェットキープ。そっと目を入れる細胞層の上にE寒天の正方形。それは、細胞の上に横たわっているとき寒天四角を移動しないでください。寒天重層することにより取り込みを改善する、ビーズと細胞との接触を増加させるために使用し、また、わずかに対物レンズの焦点面に良好それらを維持、細胞を圧縮する。 </li><li> 2時間以上毎に1分、皿にふたをする顕微鏡ステージ上に配置し、自動的に、赤、緑、位相チャンネルで写真を撮る。 </li><li>選択し、食作用の全体のプロセスが含まれている細胞事象に焦点を当てる。最適化された3チャンネルをマージし、専門的な画像処理ソフトを使ってムービーに写真を組み立てる。赤色および緑色チャネルにおいて選択された各ビーズの蛍光強度を定量化し、緑/赤の比は、細胞の動的ファゴソームROS産生を反映する。 </li></ol> 2。細胞内スーパーオキシド生産の定量的および中期スループット測定<ol><li> Oを収集NE 80％コンフルエントHL5C培地10ml中の細胞性粘菌の一品。 5分間850×gで遠心分離細胞性粘菌細胞は、慎重に、完全に余分なメディアを吸引除去する。 SS6.4バッファに再懸濁した細胞[ソレンセン·バッファ内の0.12のソルビトール（14.69のKH 2 PO 4および6.27のNa 2 HPO 4）、pHが6.4]、細胞をカウントし、6×10 6細胞/ mlの最終濃度に希釈する（議論の中で、追加のコメントを参照してください）​​。 </li><li> （議論の中で、追加のコメントを参照してください）​​白い不透明な96ウェルプレートの各ウェルに細胞懸濁液50μlを加える。 </li><li>必要に応じて、各ウェルにマルチチャンネルピペットを用いへDHEストック（DMSO中30 mM）を希釈したDHEのSS6.4、ピペット50μlと500倍に希釈します。 DHEの最終濃度は30μmである。反応がすぐに開始されます。 </li><li>刺激または阻害剤は、特定のニーズに応じて、この時点で、または他の時点で添加することができる。 </li><li> 「トップリーディング＆＃エンドポイントを使用34、522 nm/605 nmでの蛍光励起/発光の蛍光マイクロプレートリーダーのモード、1時間、2分毎に読み取り、媒体振れ22℃で5秒/分</li></ol> 3。定量的および細胞外のH 2 O 2産生のハイスループット測定<ol><li> 2.1と2.2で説明したのと同じ手順に従ってください。 </li><li> SS6.4、渦の10ミリリットルに、HRPの株式の5μL（蒸留水に100 U / ml）を加えて希釈したHRPを準備したり、よく混ぜ、後で使用するために氷の上に維持するためにチューブを反転させる。希釈したHRP溶液は0.05 U / mlである。 </li><li>それぞれ、AURストック（DMSO中10 mMのAUR）および6.2510μMの最終濃度までSS6.4バッファに希釈したHRP溶液、および0.005 U / mlのを混合することにより、AUR反応液を調製する。例として、40の反応のための反応混合物を調製する希釈したHRPを400μlとAUR原液2.5μlのSS6.4の1600μLを混ぜる。 </li><li> AURミックス50μLを追加必要に応じて、マルチチャンネルピペットを用いて各ウェルにTURE。今反応が開始されます。 </li><li>刺激または阻害剤は、特定のニーズに応じて、この時点で、または他の時点で添加することができる。 </li><li> 530 nm/590 nmで蛍光励起/発光の蛍光マイクロプレートリーダーの終点「トップ読み」モードを使用し、22℃で、1時間、中ブレ5秒/分ごとに2分を読む</li></ol>

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利害の対立が宣言されていません。

前述の方法に比べて、OBGアッセイは、動的に代えて、細胞の集団からの平均ROSシグナルを測定する、単一細胞レベルでファゴソームROS生成の過程を視覚化する。このような人口の平均化の方法は非同期食作用によって引き起こされる重要な情報を不明瞭にする傾向があります。我々が正常に細胞性粘菌にDHEとAURアッセイを適応とプロトコルを最適化した。最も重要なことは、我々はこ?...

活性酸素種（ROS）は、宿主防御、シグナル伝達、組織の発達および損傷に対する反応、ならびに高血圧症および癌などの生物学的プロセスの様々に関与している。よく研究ファゴソームNADPHオキシダーゼの機械は、好中球&#39;ファゴソーム1で摂取細菌を殺すために、酸化的バーストとして知られている急速なROS発生、に捧げられています。また、ミトコンドリア呼吸鎖の副産物としての電子の漏れが以前はROSの規制されていない元の原因であると考えられていた。しかし、最近では、マウスマクロファージ2中の細菌の死滅intraphagosomalに貢献する重要な機構として同定された。近年では、社会的なアメーバ、 細胞性粘菌は 、自然免疫応答の細胞本来のメカニズムを研究するための強力かつ人気の高いモデルとなっています。実際、 細胞性粘菌と人間の食細胞はmoleculaにおける保全の驚くほど高いレベルを共有細菌は、飲み込みを感知して、3,4を殺すための責任のR機械。このような、NADPHオキシダーゼ、カタラーゼ、スーパーオキシドジスムターゼ、などのROS産生や解毒に関わるタンパク質や酵素のホモログは、人間と細胞性粘菌の両方に記載されています。最もよく研究され、アメーバのように、 細胞性粘菌は、哺乳類のモデル系に比べていくつかのユニークな利点を提供します。それらは、8〜10時間の倍加時間で、CO 2を必要とせずに室温で成長する。それらは容易に付着または懸濁培養物として維持することができる。また、彼らの完全に配列決定し、注釈付きの半数体ゲノムに、かつ容易に遺伝子操作のおかげで、 細胞性粘菌は非常に魅力的な実験モデル生物となっています。 これまでの研究では、活性酸素による酸化後に蛍光を発する（総称OxyBurstグリーン、OBGとして知られている）フルオレセインの様々な塩素化およびフッ素化誘導体は、活性酸素のレポーターとして使用されてきた。細胞透過性EST細胞非透過性デキストラン又はタンパク質結合誘導体は、細胞外ROSを測定するために使用される一方erified誘導体は、細胞質のROSを測定するために使用される。特に、OBG BSA-コーティングされたビーズは、既に哺乳類細胞5ファゴソーム内ROS産生を検出するために使用されてきた。しかし、マイクロプレートリーダーベースのアプローチは、細胞の集団からの平均ROSの生成カーブを与えることができる。現在のプロトコルでは、 細胞性粘菌 、専門の食細胞、および最適化された実験条件を使用することにより、我々は、ビーズ上に任意のオプソニンを結合させることなく、安定的かつ効率的な食作用を得る。 DHEは、ROS産生6-9を検出するために、そのような哺乳動物好中球およびマクロファージなどの種々のモデル系において使用されている。一方、メソッド8,10の特異性と感度をある程度の論争があります。アンプレックスレッドの改良版として、AURの蛍光強度を測定することがより適してpHに対する感度が低い非中性または弱酸性的環境におけるROS。 AURは最近、いくつかの哺乳類系11,12が、非常に多くの場合、弱酸性増殖培地を必要とする非哺乳類モデルにおけるその使用に適用されている、まだ報告されていない。また、定量的かつ動的な社会アメーバ細胞性粘菌におけるROS産生および局在を測定するための公表プロトコルがありません。 プロトコルの提示組の目標は、様々なROS及びそれらの局在をモニタリングすることが容易で汎用性の高いソリューションを提供し、さらにROS関連の細胞機構への洞察を与えることである。 OBGアッセイのために、我々は、 細胞性粘菌細胞によるOBG被覆ビーズの取り込み後のファゴソームROS生成のプロセス全体を監視するために、ライブ顕微鏡を用いて、細菌のintraphagosomal死滅の機序を研究するための新しいアプローチを提供した。我々は、細胞内superoxを測定するために、 細胞性粘菌媒体スループットDHEとAURアッセイを最適化した強力なROS刺激剤としてLPSを使用して、それぞれIDEおよび細胞外のH 2 O 2の生産、、。 LPSが最近貪食細菌13に向けて細胞性粘菌の殺菌活性を増加させることが示されたことに注意してください。また、DEDTCおよびカタラーゼを用いた治療は、明示的にこれらの2つの方法を具体的に、異なる種類や細胞性粘菌におけるROSの細胞内局在を測定することを確認した。最後に、OBGアッセイは、さらに、動物細胞の食作用性受容体のリガンドでビーズをオプソニン化することにより、任意の接着性食細胞に適合させることができる。原則として、DHEおよびAURアッセイはまた、適切な実験条件下で任意の接着性または非接着性の細胞で測定用に微調整することができます。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>OxyBURST Green H2HFF BSA</td>
			<td>Invitrogen</td>
			<td>O-13291</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A-20004</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxylated silica beads</td>
			<td>Kisker Biotech</td>
			<td>PSi-3.0COOH</td>
			<td></td>
		</tr>
		<tr>
			<td>HL5C medium</td>
			<td>ForMedium</td>
			<td>HLC0102</td>
			<td></td>
		</tr>
		<tr>
			<td>LoFlo medium</td>
			<td>ForMedium</td>
			<td>LF1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>204010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihydroethidium</td>
			<td>Sigma</td>
			<td>37291-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed</td>
			<td>Invitrogen</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Roche</td>
			<td>10108090001</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyldithiocarbamate (DEDTC)</td>
			<td>Sigma</td>
			<td>D3506-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C9322-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanamide</td>
			<td>Sigma</td>
			<td>187364-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides</td>
			<td>Sigma</td>
			<td>L2630-25G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>ibidi &#956;-Dish 35 mm, high</td>
			<td>ibidi</td>
			<td>81156</td>
			<td>Bottom made of optically clear plastic</td>
		</tr>
		<tr>
			<td>MatTek dish 35 mm</td>
			<td>MatTek corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Bottom made of a glass coverslip</td>
		</tr>
		<tr>
			<td>Scepter Cell Counter</td>
			<td>Merck Millipore</td>
			<td>PHCC00000</td>
			<td></td>
		</tr>
		<tr>
			<td>White 96 well plate</td>
			<td>Nunc</td>
			<td>236108</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>SORVALL</td>
			<td>Legend RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy Mx</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting, visualizing and quantitating the generation of reactive oxygen species in an amoeba model system

活性酸素種（ROS）のいずれかを動的に触媒的または自然に相互変換又は排除される反応性短命の、酸素含有分子の範囲を含む。最もによるROS及びそれらのソースおよび細胞内局在の多様性の短い寿命のために、全体像は、プロトコルの組み合わせを用いて注意深く測定することによって得ることができる。ここでは、OxyBurstグリーン（OBG）を使用して、三つの異なるプロトコルのセットをコーティングしたビーズ、またはジヒドロエチ（DHE）を提示し、アンプレックスUltraRed（AUR）は、定性的および定量的な細胞性粘菌などの専門食細胞内の様々な活性酸素を監視すること。我々は手順をコーティングビーズを最適化し、動的単一細胞レベルでintraphagosomal ROS生成を可視化するOBGコートビーズとライブ顕微鏡を使用していました。我々は、 大腸菌からリポ多糖（LPS）を同定細胞性粘菌におけるROS生成のための強力な刺激として大腸菌 。また、我々は、dはリアルタイム定量的にそれぞれ、細胞内および細胞外スーパーオキシドH 2 O 2産生を測定するためDHEとAURを用いて媒体スループットアッセイをeveloped。

ファゴソーム内ROSの発生を顕微鏡（ ファイルS1）により定性的にかつ動的に視覚化することができる。アレクサフルオロ594により放出された赤色の蛍光はpH非感受性であり、OBGの酸化はグリーンチャンネルでその蛍光を増加させながら、ファゴソーム環境では一定のまま。 インビトロでの発光スペクトルは、酸化されて非酸化OBG被覆ビーズは、酸化後の強度の有意な増加?...

Watch this Scientific Journal Video about Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System at JoVE.com

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

我々は、定性的および定量的研究のための様々なアメーバおよび哺乳動物細胞モデルに適用することができる反応性酸素種（ROS）の測定のためのプロトコルのセットを適合。

、検出し可視化し、アメーバモデル系で活性酸素種の生成を定量

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

detecting-visualizing-quantitating-generation-reactive-oxygen-species

Research

JoVE Journal

Biology

16.0K ビュー.  University of Geneva. 我々は、定性的および定量的研究のための様々なアメーバおよび哺乳動物細胞モデルに適用することができる反応性酸素種（ROS）の測定のためのプロトコルのセットを適合。 

ビデオ: Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

の心臓の鼓動の可視化ショウジョウバエ</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

生物学

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 ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

細胞侵襲DISC1タンパク質種の生成、精製とキャラクタリゼーション

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

モデルシアノバクテリア種のマークとマーカーレス変異体の生成

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

心筋パフォーマンスと酸素消費量の研究のための齧歯類のワーキングハートモデル

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

食作用の初期段階の可視化

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

酸性細胞外 pH 培養系の確立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

切り替え可能なプロトポルフィリンIX依存性活性酸素種のin situ光生成によるマウス皮膚における幹細胞ニッチの刺激と組織再生

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...