タバコbenthamiana植物は25℃での温度制御成長チャンバーで栽培されています。 3月6日週令の植物の完全に展開された健康な葉が使用されます。 ヒント：より良い結果が新たに出現した葉を使用して得られる 1。アグロバクテリウム一時的な浸透のプロトコル： 日1 <ol><li>細胞死のために、アッセイする遺伝子（群）とを含むベクターを用いて適切なベクターを含むアグロバクテリウムツメファシエンス（LBA4404株）のグリセロールストックを持つストリークLB /リファンピシン（25μg/ mlの）/カナマイシン（100μg/ ml）を寒天プレート構成的プロモーターの下でGUSカセット。常にネガティブコントロールとして空のベクターコントロールが含まれています。 </li><li> 28℃で2日間インキュベートする。 </li></ol>日3 <ol><li>以前に縞各LB /リファンピシン/カナマイシンプレートから単一コロニーとリファンピシン（25μg/ mlの）とカナマイシン（100μg/ ml）を含むLB 2 mlを接種する。 </li><li>最大成長の密度に達するまで、200rpmで24時間28℃で振盪することにより、各培養液を、。 </li></ol> ヒント：クランピングは、時々ケースの文化は徹底的に進める前に（ピペッティングすなわち）再懸する必要があるで、観察される。 日4 <ol><li>インキュベーションの後、新鮮なLB（適切な抗生物質を含む）とアセトシリンゴン（最終濃度25μM）で10 mLまでの総容積を増加させる。 </li><li>別の16時間28℃で振盪することにより、各培養液を、。 </li></ol>日5 <ol><li>翌日は、室温で10分間4000 × gで（アセトシリンゴンを追加しない）浸潤の培地（10 mMのMgSO 4を 7H 2 O、9mmのMES、pHは5.6）と遠心分離を10 mlを加えることによって二度、それぞれの文化を洗う。 </li><li>最後の遠心分離工程の後に、アセトシリンゴン（100μM）を含む5mlの浸透培地中でそれぞれの文化を再懸濁します。 </li><li> OD 600nmのを測定し、0.1と0.9の間で測定値が許容範囲です。 ヒント：0.1と低いのOD 600nmのは問題もなく使用されている、しかし、より高いODSは、より一貫性のある結果を生むかもしれません。 </li><li>感染症のためのアグロバクテリウム培養液を準備するより3時間培養をインキュベートする。 </li><li>分析する遺伝子（群）とGUSカセットを含む培養と1:1の比率で、ネガティブコントロール（アグロバクテリウムは、空のベクターを含む）を含む混合培養。 </li><li> 1mlの無針注射器（図1）を使用して新興の葉の背軸（下）側に潜入。 ヒント：ネガティブコントロールの混合物（空のベクトル+ GUSカセット）と同じ植物の反対側の葉の上に（遺伝子X + GUSカセット）、アッセイする遺伝子を含有する混合物の両方に潜入。それぞれの治療のために三重工場を使用してください。 </li><li> 3日後の浸潤の消費税は、GUS蛋白質の発現のために葉やアッセイに潜入。 </li></ol> 2。組織化学的GUSアッセイ日8 <ol><li>切除した葉にX - gluc基板媒体に潜入掃除機。 </li><li>室温で一晩、または個別の青色染色が表示されるまで、暗闇の中でインキュベートする。 </li></ol>日9 <ol><li>蒸留水ですすいでください。 </li><li>クロロフィルが削除されるまで、再び蒸留水に転送し、70％エタノールでインキュベートする。視覚的にGUSの発現レベルを評価する。 </li></ol> 3。蛍光MUGアッセイ： 日8 <ol><li>液体窒素を使用して、すり鉢で浸透した葉ディスクを粉砕することにより総タンパク質を分離します。 GUS抽出バッファー（50mMのNAPI液（pH7.0）、10mMのEDTA、0.1％トリトンX - 100、0.1％N -ラウロイルサルコシンナトリウム塩、β-メルカプトエタノール（0.7μL/ ml）を氷上でサンプルを保ちながらの100μLのを追加。 </li><li> 4℃で5分間、14,000 gでサスペンションを遠心し、全可溶性タンパク質として上清を取る。 </li><li>製造元の指示に従って光度計を使用して濃度を測定します。 </li><li> GUS抽出バッファーを使用して、各サンプルの総可溶性タンパク質100μgのためにタンパク質の濃度を調整します。 </li><li> 2 mMの最終濃度にGUS抽出緩衝液中で調製MUG基板（4 - メチルウンベリフェリル-β- D -グルクロニド三水和物）を追加します。サンプルあたり100μL（タンパク質+マグの基板）の合計量は十分なはずです。 </li><li> 37℃で1時間反応をインキュベート℃に</li><li> GUSストップバッファー（0.2 M Na 2 CO 3）を800μlを添加して反応を停止します。 </li><li>蛍光が365 nmと455 nmの発光波長の励起波長でプレートリーダーを使用してMU標準曲線に値を比較測定します。蛋白質/分nmolのMU /μgの全可溶性としてGUS活性のレベルを報告する。 </li></ol> 4。株式とソリューション： <ol><li>アセトシリンゴン（1 M）株式（FW = 196.2グラム） </サブ&gt; - DMSO 5mL中0.981グラム</li><li>カナマイシン（100 mg / ml）を株式には、 -のdH 2 0で準備する-フィルター滅菌</li><li>リファンピシン（25 mg / ml）の株価は - DMSOで準備</li><li>ルリア - ベルターニ（LB）液体増殖培地：（1L） 1％（w / v）のバクト - トリプトン、10gの 0.5％（w / v）のバクト酵母エキス、5gの 170 mM塩化ナトリウム10グラム pHは - 7 </li><li>アセトシリンゴン（25μM）を1mlの最終容量の1Mストックの=25μlの</li><li>浸透ミディアム：（1L） 10mMのMgSO 4を 0.7のdH 2 0（FW = 246.48グラム）、2.4648グラム 9mmのMES（FW = 213.25グラム）、1.91925グラム pH値 - 5.6 </li><li>アセトシリンゴン（100μM）を1mlの最終容量の1M株式=100μlの</li><li> GUS抽出バッファー 50mMのNAPI液（pH7.0）、10mMのEDTA、0.1％トリトンX - 100、0.1％N -ラウロイルサルコシンナトリウム塩、β-メルカプトエタノール（0.7μL/ ml）を</li><li> 2倍のリン酸緩衝液 0.2MのNaH 2 PO 4、0.2 MのNa 2 HPO 4 pHを7 </li><li> X - Gluc基質溶液 0.1mlのメタノール5 - ブロモ-4 - クロロ-3 - インドリルBD -グルクロニド（X - Gluc）1mgを溶解する。 、20μlの0.1Mフェリシアン化カリウム、10μlのトリトンX - 100を10％、蒸留水850μlを1ミリリットル2倍のリン酸緩衝液を追加します。 </li></ol> 5。代表的な結果： 細胞死が発生したときに、このプロトコルに続いて、GUSの発現の消失が期待されている。我々は同時にCYTO -保護シロイヌナズナのBcl - 2関連athanoGene（BAG）ファミリーのメンバーを持つβ-グルクロニダーゼレポーター遺伝子、（GUS）を発現している。我々の共同浸潤N. benthamianaは、アグロバクテリウム LBA4404株を用いて35S駆動型BAGの発現カセットとGUSベクターをpCAMBIA 2301を残します。図2に示すように、GUS染色で可視増加は、この浸潤後に観察された。逆に、Bcl - 2ファミリーの既知のアポトーシスメンバーBAXが使用された時、GUS染色の著しい減少は、（図2）が観察された。どちらの場合でも、GUS発現は、コントロールに比べて目に見えて違っていた。しかし、表現の差は少ないが明らかであるときは、蛍光MUGアッセイは、GUS発現をquanitateに行うことができます。 <img src="/files/ftp_upload/2680/2680fig1.jpg" alt="図1"/> 図1。 Nの背軸側のアグロバクテリウムの混合物の浸透の例benthamianaは、1mlの無針注射器を使用して残します。 <img src="/files/ftp_upload/2680/2680fig2.jpg" alt="図2"/> 図2。 GUSのベクトルはNで共発現されたbenthamianaは抗死の遺伝子、および細胞死の既知の誘導剤で残します。 GUSのレベルは空のベクトル制御（GUSコントロール）と比較した。

<ol>
	<li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUS+fusions:+,+&#946;-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">GUS fusions: , &#946;-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3901 (1987).</li><li>Nishihara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+&#946;-Glucuronidase+Gene+in+Pollen+of+Lily+(Lilium+longiflorum),+Tobacco+(Nicotiana+tabacum),+Nicotiana+rustica,+and+Peony+(Paeonia+lactiflora)+by+Particle+Bombardment.">Expression of the &#946;-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.</a> Plant Physiol. 102, 357-357 (1993).</li><li>Hodal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 87, 115-115 (1992).</li><li>Kabbage, 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+BAG+proteins:+a+ubiquitous+family+of+chaperone+regulators.">The BAG proteins: a ubiquitous family of chaperone regulators.</a> Cell Mol. Life Sci. . 65, 1390-1390 (2008).</li><li>Kang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtBAG6,+a+novel+calmodulin-binding+protein,+induces+programmed+cell+death+in+yeast+and+plants.">AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.</a> Cell Death Differ. 13 (1), 84-84 (2006).</li><li>Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 165 (1), 1-1 (2006).</li><li>Takayama, S., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+targeting+and+regulation+by+BAG+family+proteins.">Molecular chaperone targeting and regulation by BAG family proteins.</a> Nat Cell Biol. 3 (10), 237-237 (2001).</li><li>Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+analysis+of+BAG-1:+a+novel+Bcl-2-binding+protein+with+anti-cell+death+activity.">Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity.</a> Cell. 80 (2), 279-279 (1995).</li><li>Vitha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+&#946;-glucuronidase+assay+in+transgenic+plants.">Quantitative &#946;-glucuronidase assay in transgenic plants.</a> Biol. Plant. 35, 151-151 (1993).</li><li>Otha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+Expression+in+Tobacco+of+a+&#946;-Glucuronidase+(GUS)+Reporter+Gene+Containing+an+Intron+Within+the+Coding+Sequence.">Construction and Expression in Tobacco of a &#946;-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence.</a> Plant Cell Physiol. 31, 805-805 (1990).</li></ol>

利害の衝突は宣言されません。

<p class="jove_content"&gt;これは、哺乳類のシステムでは一般的な植物で細胞死の検出技術を使用することは困難です。 GUSレポーターシステムと組み合わせることで、我々は細胞死のプレイヤーの検出と解析のための植物ベースの、敏感な手法を提案する。このメソッドは、生きている細胞が発生するGUSの発現に必要であるという単純な事実を利用しています。意味のある結果と再現性を確保するためには、GUSのカ...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>リファンピシン</td><td> VWR </td><td> IC19549001 </td><td> DMSOに25mg/mLの株式</td></tr><tr><td> 4&#39; -ヒドロキシ-3&#39;、5&#39; -ジメトキシ（アセトシリンゴン） </td><td> VWR </td><td> TCD2666 </td><td> DMSOの0.981グラム/ mLの（1M株） </td></tr><tr><td> 2 - （4 - モルホリノ）エタンスルホン酸一水和物（MES） </td><td> VWR </td><td> EM - 6110 </td><td>浸透培地1 Lを作るための水の1.92グラム/ L </td></tr><tr><td>バクトトリプトン</td><td>フィッシャー</td><td> BP1421 - 2 </td><td> LB培地1 Lを作るための水で10g / L </td></tr><tr><td>バクト酵母エキス</td><td> VWR </td><td> EM1.03753.0500 </td><td> LB培地1 Lを作るための水に5グラム/ L </td></tr><tr><td>ナトリウム塩化</td><td> VWR </td><td> EM - 7710 </td><td> LB培地1 Lを作るための水で10g / L </td></tr><tr><td>ナトリウム一塩基性リン酸一水和物</td><td> VWR </td><td> MK - 7868〜12 </td><td>バッファNAPIの50mmの作るための水に2.5グラム/ L </td></tr><tr><td>リン酸ナトリウム、二塩基七水和物</td><td> VWR </td><td> EMD - SX0715 - 1 </td><td>バッファNAPIの50mmの作るための水に5グラム/ L </td></tr><tr><td>硫酸マグネシウム七水和物</td><td> VWR </td><td> EM - MX0070 - 1 </td><td>浸透培地1 Lを作るための水では2.5 g / Lの</td></tr><tr><td> N -ラウロイルサルコシン</td><td> VWR </td><td> TCL0151 - 500G </td><td> GUS緩衝液で0.1％v / vの</td></tr><tr><td> 5 - ブロモ-4 - クロロ-3 - インドキシル-β- D -グルクロニドシクロヘキシル塩（X - gluc） </td><td>ゴールドのバイオテクノロジー</td><td> G1281C </td><td>メタノール100％の1mg/100uL </td></tr><tr><td> 4 - メチルウンベリフェリル-μ- D -グルクロニド水和物（MUG） </td><td>シグマ</td><td> M5664 </td><td> GUS抽出バッファー100μlの2 mMの</td></tr><tr><td>フェロシアン化カリウム三水和物</td><td> VWR </td><td> EM - PX1460 - 1 </td><td> X - glucの基質溶液のための100mMの株式</td></tr><tr><td> β-メチルウンベリフェロン（MU） </td><td>シグマアルドリッチ</td><td> M1381 </td><td> MU検量線の使用GUS抽出バッファー用</td></tr><tr><td>ナトリウム炭酸</td><td> VWR </td><td> EM - SX0395 - 11 </td><td> GUSストップバッファを作るための水に0.2 M </td></tr></tbody></table>

a &#946;-glucuronidase (gus) based cell death assay

我々は、同時に細胞死の推定正または負の調節で、レポーター遺伝子、β-グルクロニダーゼ（GUS）を発現する新規の一時的な植物発現システムを開発した。このシステムでは、N. benthamianaの葉を分析する遺伝子を含む35S駆動発現カセットと同じ場所に浸潤、およびGUSベクターpCAMBIA 2301車両としてアグロバクテリウム株LBA4404を使用しています。生きている細胞が発生するGUSの発現に必要とされているため、このマーカー遺伝子は、細胞死の正の調節因子と共発現されると、GUS活性の損失が予想されます。同様に、GUS活性は、抗アポトーシス遺伝子をベクターコントロールと比較して使用する場合に観察される増加した。以下に示すように、我々が正常にプロと抗死のプレーヤーの両方を分析するために私たちのラボでこの制度を利用しています。これらは、植物の抗アポトーシスBcl - 2の関連athanoGene（BAG）家族だけでなく、そのようなBAXのような細胞死の既知の哺乳類の誘導を、含まれています。さらに、我々はこれらのタンパク質の可能な翻訳後修飾/アクティベーションについての手がかりを提供することができるタンパク質内の特定の切り捨て、の死の機能を解析するためにこの制度を利用しています。ここで、我々は、特定の遺伝子の死の機能を検討する上で最初のステップとして、迅速かつ高感度の植物ベースの手法を提案する。

Watch this Scientific Journal Video about A Î²-glucuronidase GUS Based Cell Death Assay at JoVE.com

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

一般的にこのようなDNAラダーやTUNELアッセイのような哺乳類のシステムで使用されるプログラム細胞死のアッセイは、しばしば植物に再現することは困難です。 GUSレポーターシステムと組み合わせることで、我々は特定の遺伝子の潜在的な死の特性を解析するために迅速、植物ベースのトランジェントアッセイを提案する。

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 ...

a-glucuronidase-gus-based-cell-death-assay

Research

JoVE Journal

Biology

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

27.9K ビュー.  Texas A&M University. 一般的にこのようなDNAラダーやTUNELアッセイのような哺乳類のシステムで使用されるプログラム細胞死のアッセイは、しばしば植物に再現することは困難です。 GUSレポーターシステムと組み合わせることで、我々は特定の遺伝子の潜在的な死の特性を解析するために迅速、植物ベースのトランジェントアッセイを提案する。

ビデオ: A &#946;-glucuronidase GUS Based Cell Death Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

ルシフェラーゼベースのレポーター遺伝子アッセイで膜の生合成を研究して

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

生物学

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

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

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 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 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

リシン毒素チェーン (RTA) 酵母の人身売買に必要な細胞成分を識別するために簡単な蛍光レポーターの試金

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

SNARE人身売買を用いた循環型の内視鏡分析法

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

比色細胞ベースのアッセイを用いてAAVに対する中和抗体を検出するためのステップバイステップ法

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

ミツバチ組織における組織学の基礎と細胞死検出

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

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 ...

連続イメージングによる細胞死開始の定量化

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

著者スポットライト:過敏反応における細胞死開始を研究するための合理化されたアプローチ 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

デジタル顕微鏡を用いた細胞死開始の研究

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...