この研究は、カナダ自然科学（NSERC）研究助成金のエンジニアリング評議会とDRBにアルバータ州農業資金コンソーシアムの助成金によって支えられている。 AMRは、NSERCヴァニエカナダ大学院奨学金、アルバータ大学の授業助手職とクイーンエリザベスII大学院の奨学金によってサポートされています。

この手順は、500μLシリコナイズチューブまたは6 mLのポリスチレン丸底FACSチューブで行うことができます。他の手順と組み合わせて実行可能性分析を行う際に、後者は通常使用されます。与えられたすべてのボリュームは、6 mLのポリスチレン丸底FACSチューブ用です。 500μLシリコナイズチューブの場合は、1 / 5ですべてのボリュームを減らす。 フローサイトメトリー分析のための最適濃度は200μLボリューム2〜4 × 10 6個の細胞です。細胞の損失は、この手順からなることがありますので、我々は、各サンプルは、プロシージャの開始時に4 × 10 6個の細胞から構成されることをお勧めします。 1。細胞の準備<ol><li>収穫の細胞は - 対応する細胞株または初代細胞アイソレーションのための具体的な手順を参照してください。 </li><li> 10分間、335 × gでサンプルを遠心し、上清をデカントする。 </li><li> 2 mLの1 ×リン酸に再懸濁し細胞（PBS）緩衝生理食塩水- / - （無カルシウム、マグネシウムがない）。 </li><li> 10分間、335 × gでサンプルを遠心し、上清をデカントする。 </li><li> 1 mLの1 ×アネキシンV結合バッファーで細胞を懸濁します。 </li><li> 10分間、335 × gでサンプルを遠心し、上清をデカントする。 </li><li> 100μLの1 ×アネキシンV結合バッファーに再懸濁し細胞。 </li></ol> 2。アネキシンV / PIのアプリケーションでは、染色<ol><li>メーカーの推奨に従ってアネキシンVを追加する[例えば、5μLアネキシンVのAlexa Fluor ® 488（Molecular Probes社、A13201）]。 </li><li>室温で15分間暗所でチューブをインキュベートする。 </li><li>各反応チューブに1 ×アネキシンV結合バッファー100μLを加える。各チューブに約200μLがあるはずです。 </li><li> 1 ×アネキシンV結合バッファー（10μL1 ×アネキシンV結合バッファーを持つ、すなわち1μLPI）で1:10に希釈されたPIの4μL（Sigma、カタログ＃P - 4864 - 10ML）を追加します。これは、各サンプルに2μg/ mLの最終的なPIの濃度が得られます。 </li><li>室温で15分間暗所でチューブをインキュベートする。 </li><li>細胞を洗浄するために、500μLの1 ×アネキシンV結合バッファーを追加します。 </li><li> 10分間、335 × gでサンプルを遠心し、上清をデカントする。 </li><li> 500μL1 ×アネキシンVの結合緩衝液と1％のホルムアルデヒド（固定）ソリューションを作成するために500μLの2％ホルムアルデヒドで再懸濁し、細胞。穏やかなフリックでチューブを混ぜる。 </li><li>氷上で10分間サンプルを修正。また、サンプルは4℃で一晩保存することができます℃で暗所で。後者の方法が選択された場合、アネキシンV / PI（補償のコントロールを含む）で標識されたすべてのチューブ全体で一貫していることを確認します。 </li><li>各サンプルへとフリックにより穏やかに混合- / - 1 mLの1 × PBSを追加。 </li><li> 8分間425 × gでチューブを遠心し、上清をデカントする。 </li><li>手順2.10と2.11を繰り返します。 </li><li>チューブをフリックしてペレットを再懸濁します。 </li><li>濃度50μg/ mlの最終濃度になるように1:100希釈のRNase A（シグマ、R4642）の16μLを追加。 37℃で15分間インキュベート℃に</li><li> / - - 1 mLの1 × PBSを追加し、フリックにより穏やかに混合する。 </li><li> 8分間425 × gでチューブを遠心する。 </li><li>現在、サンプルを分析する準備が整いました。アネキシン/ PI染色は他の手続きと並行して実行されている場合、あるいは、試料は、その後の染色工程のために使用することができます。 </li></ol> 3。代表的な結果： 偽陽性のPI染色の度合いをした細胞タイプと細胞株の例を図1に示されています。偽陽性のイベントを考慮して、細胞を固定してから（図2B）RNase処理を受けていない細胞と比較して偽陽性のイベント、数の有意な減少でのRNase Aでこの結果を処理した。図2CはRNase処理を持たない細胞に比べて、RNase処理を受けた細胞の代表画像を示す。図3は、固定およびRNase処理のステップで変更されたアネキシンV / PIプロトコルは、偽陽性染色のイベントの数を制限することにより、従来のプロトコルを改良した方法を示しています。 <img src="/files/ftp_upload/2597/2597fig1.jpg" alt="図1" /> 図1。従来のヨウ化プロピジウム染色のプロトコルは、偽陽性のイベントのかなりの数につながる。我々は以前に動物モデルの広範なだけでなく、細胞株10の様々な主要な細胞間の偽陽性染色の程度を評価した。代表的な画像は、3つのユニークな細胞集団（ - RAWマクロファージおよびつの主要細胞 - マウス骨髄マクロファージ（BMM）と金魚の主腎マクロファージ（PKM）1つの一般的に使用される細胞株）における偽陽性染色の程度を示す。真陽性のイベントが核コンパートメント内でPIとDRAQ5の間に予想される共局在を（黄色の領域は、PIとDRAQ5の間で共局在を表す）が表示されます。 DRAQ5は非常に体肢です。NE透過性染料その生細胞と死細胞10,11,12の両方で正確に汚れ核コンパートメント。共局在DRAQ5汚れを簡単に視覚的な決定のための緑のpseudocolourを与えられた。 <img src="/files/ftp_upload/2597/2597fig2.jpg" alt="図2" /> 図2。 PI核染色の精度を向上させる 。 （A）我々はDRAQ5に十分に特徴付けられた核染色（BrdUを、7 - AADとDAPI）の数の類似度に基づいて、PI核染色の精度を評価した。のBrdUは増殖細胞のDNAに組み込まれ、そのようにのみ核コンパートメント内に染色される合成ヌクレオシドである。 7 - AADは、PIと同様のDNAと、親和性の高い、無傷の原形質膜を越えることはできません。 DAPIは、DNAに強い親和性を有しており、蛍光顕微鏡で染色核の最も一般的に使用されています。類似度は正確にRAW 264.7マクロファージにおける細胞核を染色する能力を強調し、BrdUを、7 - AAD、DAPIとDRAQ5との間で&gt; 99％であった。対照的に、従来のプロトコルに基づいて細胞質PI染色はDRAQ5に相対的染色のパーセント類似性の著しい低下につながる。マウス骨髄マクロファージ（BMM）と金魚の主腎マクロファージ（PKM）：（B）も同様の結果が試験された2つの主要な細胞で観察される。染色手順内の特定の段階では50μg/ mlのRNaseの混入は非核兵器PI染色を削除し、大幅にDRAQ5染色の相対類似度を増加させる。 （C）一次性腎マクロファージの代表的な画像は、RNase処理がある場合とない場合のセルの類似度を示す。治療法の違いを簡単に視覚的判定のために、DRAQ5は緑色を与えられた。黄色の領域は、BrdUとDRAQ5、およびPIとDRAQ5の間で共局在を表しています。 <img src="/files/ftp_upload/2597/2597fig3.jpg" alt="図3" /> 図3。修正されたアネキシンV / PIは大幅に偽のアポトーシス/壊死性イベントを減少します。主な金魚の腎臓のマクロファージ（PKM）は、従来と変更されたアネキシンV / PIプロトコールで染色した。散布図はアネキシンV / PIは、金魚のPKM​​に染色描く。左の散布図は、PKM細胞の従来のアネキシンV / PI染色（無RNaseを）示しています。右上の散布図は、変更されたアネキシンV / PIプロトコル（RNaseの）で染色PKM細胞を示しています。偽陽性の汚れの除去に起因するPI染色の顕著な減少中のRNaseの結果の追加。 PKM細胞は、培養液、初期の前駆細胞（EP）、単球（モノ）と成熟し​​たマクロファージ（マットMAC）内の異なる個体群に基づいて分析した。偽陽性のイベントの除去に起因する正のPIのイベント数の減少は、より小さな早期の前駆細胞に比べて大規模な成熟したマクロファージ細胞においてより顕著である。従来のプロトコルに基づいて結論が誤って、より成熟したマクロファージサブセットの細胞死に正の相関を示唆しているでしょう。

<ol>
	<li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J Immunol Methods. 184, 39-51 (1995).</li><li>Vermes, I., Haanen, C., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+apoptotic+cell+death.">Flow cytometry of apoptotic cell death.</a> J Immunol Methods. 243, 167-190 (2000).</li><li>Cornelissen, M., Philippe, J., De Sitter, S., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+expression+in+apoptotic+peripheral+blood+lymphocytes:+An+electron+microscopic+evaluation.">Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation.</a> Apoptosis. 7, 41-47 (2002).</li><li>Fried, J., Perez, A. G., Clarkson, B. 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A., Lassota, P., Traganos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+apoptotic+cells+measured+by+flow+cytometry.">Features of apoptotic cells measured by flow cytometry.</a> Cytometry. 13, 795-808 (1992).</li><li>Kroemer, G., Dallaporta, B., Resche-Rigon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+death/life+regulator+in+apoptosis+and+necrosis.">The mitochondrial death/life regulator in apoptosis and necrosis.</a> Annu. Rev. Physiol. 60, 619-642 (1998).</li><li>Denecker, G., Vercammen, D., Declercq, W., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+and+necrotic+cell+death+induced+by+death+domain+receptors.">Apoptotic and necrotic cell death induced by death domain receptors.</a> Cell Mol. Life Sci. 58, 356-370 (2001).</li><li>Faleiro, L., Lazebnik, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+disrupt+the+nuclear-cytoplasmic+barrier.">Caspases disrupt the nuclear-cytoplasmic barrier.</a> J Cell Biol. 151, 951-959 (2000).</li><li>Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., Barreda, D. 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利害の衝突は宣言されません。

アネキシンV / PIプロトコルは、従来のプロトコルの改良版であると考慮もPIに対して高い親和性を持つ細胞質のRNAの存在を取るここで紹介。染色手順の後半で1％ホルムアルデヒド固定ステップの後のRNase A（50μg/ mLの）の導入は、核PI染色の精度を大幅に向上します。負の効果は核PI染色や細胞膜アネキシンV染色で観察されていません。 RNase A処理することなく、PI染色結果の最大40％のカスタマーが、下流の結論10日に潜在的に有意かつ負の影響につながる偽陽性のイベントにつながる可能性があります。 、変更したアネキシンV / PI染色プロトコルは単純で、細胞の種類（ プライマリセルの広い範囲で効果的に使用されています：マウス骨髄マクロファージおよび脾細胞;豚肺常駐細胞、肺胞マクロファージ、腸間膜リンパ節の分離株、末梢血白血球、脾細胞、ニワトリ胚盤葉細胞を、金魚の主腎マクロファージ、コイ末梢血白血球、 細胞株 ：RAW 264.7マクロファージ、Jurkat T細胞、豚3D4/31マクロファージ、CCL - 71金魚のひれの繊維芽細胞、3B11ナマズのB細胞10）。細胞は差初代細胞は一般的に偽陽性のイベント10の大きい番号を持つとともに、偽陽性のPI染色によって影響されることに注意することは重要です。これらの細胞集団間の違いの偽陽性PI染色が部分的にセルサイズの違いに起因することがありますが、またRNAの含有量の違いから発生する可能性があります。この手順のような、組み込みは、他の人の間で利用する実験系、、遺伝毒性ストレスを受けた細胞は、細胞周期停止のようなチミジンやヒ​​ドロキシ尿素などの薬剤、ウイルス感染細胞、および発達進行胚細胞の研究で処理した細胞に特に関連として細胞のRNA合成における離散的な変化によって特徴付けられる。

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		<th>Material Name</th>
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<td>1 x PBS-/-</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 x Annexin V Binding Buffer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556454</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-Alexa Fluor 488</td>
<td>&nbsp;</td>
<td>Molecular Probes (Invitrogen)</td>
<td>A13201</td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide (PI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
<td>1 mg/mL in H2O</td>
</tr>
<tr>
<td>2% Formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F1268</td>
<td>&nbsp;</td>
<tr>
<td>RNase A from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R4642</td>
<td>&nbsp;</td>
<tr>
<td>DRAQ5</td>
<td>&nbsp;</td>
<td>Biostatus</td>
<td>DR50050</td>
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modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death

細胞のアポトーシスの研究は著しく、フローサイトメトリーベースのメ​​ソッドの導入以来、影響されています。ヨウ化プロピジウム（PI）は広く細胞が原形質膜の完全性と透過性の1,2の違いによって、実行可能なアポトーシス、または壊死性かどうかを判断するためにアネキシンVと組み合わせて使用されます。アネキシンV / PIプロトコルでは、アポトーシス細胞3を研究するための一般的に使用されるアプローチです。それは細胞が4,5住んで染料を除外するその能力に基づいて、経済的安定と細胞生存率の良い指標であるため、PIは、より頻繁に他の核染色より使用されています。セルを入力するPIの能力は、膜の透過性に依存しています。PIは、無傷の原形質膜1,2,6の存在によるライブや早期のアポトーシス細胞を染色していません。後期アポトーシスとネクローシス細胞では、プラズマと核膜の完全性は、PIは、膜を通過する核酸にインターカレートし、赤色蛍光1,2,9を表示できるように、7,8を減少させます。残念ながら、我々は従来のアネキシンV / PIプロトコルが細胞質コンパートメント10内にRNAのPI染色に関連付けられている偽陽性のイベント（最大40％）のかなりの数、につながることがわかります。最高発生10を示す：（細胞質比が&lt;0.5核）初代培養細胞や動物モデルの広い範囲の細胞株は大型細胞で、影響を受けます。ここで、我々は、従来の方法に比べて細胞死の評価のための有意な改善を提供する修正アネキシンV / PI法を示しています。このプロトコルは、染色後の細胞に含まれるRNaseのエントリーを促進するための固定細胞中に細胞透過性の変化を利用しています。タイミングとRNaseの濃度の両方が細胞質RNAの除去に最適化されています。その結果、従来のアネキシンV / PIプロトコル（細胞質PI染色で&lt;5％のイベント）に比べて大きな改善です。

Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

細胞死の評価のための正確な方法が記載されている。プロトコルは、細胞株および動物モデルの広い範囲からの一次細胞は40％、偽陽性のイベントを表示する従来のアネキシンV /ヨウ化プロピジウム（PI）のプロトコル、改良したものです。

細胞死の正確な評価のために修飾されたアネキシンV /ヨウ化プロピジウムのアポトーシスアッセイ

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

modified-annexin-vpropidium-iodide-apoptosis-assay-for-accurate

Research

JoVE Journal

Biology

102.4K ビュー.  University of Alberta. 細胞死の評価のための正確な方法が記載されている。プロトコルは、細胞株および動物モデルの広い範囲からの一次細胞は40％、偽陽性のイベントを表示する従来のアネキシンV /ヨウ化プロピジウム（PI）のプロトコル、改良したものです。

ビデオ: Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

心原性誘導効率を定量化するために修飾されたマウス胚性幹細胞ベースのアッセイ

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

生物学

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

β-グルクロニダーゼ（GUS）に基づく細胞死アッセイ

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

シトクロムの細胞質マイクロインジェクションによるアポトーシスの活性化 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

胚におけるプログラム細胞死を検出するためのLysoTrackerの使用は、胚性幹細胞を分化

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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, ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

トラッキングアナスタシス、アポトーシスを逆に細胞生存現象のための戦略

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

ポリソームプロファイリングによる哺乳動物細胞における選択的mRNAの翻訳の評価

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

ヨウ化黄色蛍光タンパク質ギャップ結合細胞間コミュニケーション アッセイ

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

成人筋幹細胞機能評価のための単一筋繊維培養アッセイ Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

CLON-Gおよび in vitro 自然死アッセイによる好中球の寿命延長

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...