Name: detection and isolation of apoptotic bodies to high purity
Uploaded: 2018-08-12T15:00:00.000+00:00
Duration: 12 min 17 s
Description: Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

I.K.H.P. に国立保健医学研究評議会 (GNT1125033 ・ GNT1140187) とオーストラリアの研究評議会 (DP170103790) からの助成金によって支えられた仕事

1. アポトーシスの誘導
<ol><li>5 分間 300 x g でセルのサンプルを遠心し、任意の既存の細胞の残骸を削除する上澄みを廃棄します。 注: 付着性のセルを使用して、事前に細胞を播くし、アポトーシス誘導前にリン酸緩衝液 (PBS) × 1 で洗います。</li>
	<li>セルの数を決定し、細胞を収集します。 注: 測定後分離に応じてお勧めします少なくとも 1 x 10 の開始セル番号7セル。</li>
	<li>1 x 106セル/mL の最終的な集中の完全なメディア (50 μ G/ml ストレプトマイシンの混合物、50 IU/mL ペニシリン (巻/巻) 10% 牛胎児血清を含むそれぞれの媒体) に再懸濁します。</li>
	<li>分注 ~ 6 ウェル プレートのウェルあたり 10 の6セル × 2。</li>
	<li>アポトーシスを誘導して、板蓋を取り外して 150 mJ/cm2の紫外線照射器を使用して細胞を照射します。これは、約 30-60 秒を取る必要があります。 注: 照射前に '非導入' セル コントロール ~0.5 x 106セルが保存されることを確認します。 注: 抗 Fas または血清飢餓6など他の方法を介してアポトーシスを誘導もことができます。</li>
	<li>37 ° c、5% CO2セル行に応じて、2-8 時間孵化させなさい。</li>
	<li>ベンチ トップ光学顕微鏡を使用して、ブレブ、apoptopodia と ApoBD 層などのアポトーシスの形態の存在を確認する細胞を可視化します。 注: 40 倍の倍率は、十分です</li>
	<li>P1000 ピペットを使用して、ピペットし、アポトーシスのサンプルを収集します。</li>
	<li>1x PBS で洗浄して最大収量を確保するため残りのサンプルと組み合わせます。</li>
	<li>収集 ~1/10th '全体アポトーシス サンプル' (WAS) を制御します。</li>
	<li>「未処理」サンプルを収集します。</li>
	<li>6 分間 3,000 x g で WAS と非導入の両方のサンプルを遠心します。</li>
	<li>1 mL の 1x PBS に再懸濁します、氷の脇します。</li>
	<li>ApoBD 分離、ステップ 2 または残りのアポトーシス サンプル 3 のいずれかに進みます。</li>
</ol>2 FACS による ApoBD 分離
<ol><li>6 分間 3,000 x g で全体のサンプルを遠心します。</li>
	<li>ペレットを中断させることがなく上澄みの大半を削除します。</li>
	<li>A5-FITC の 75 μ L と 1 x 10 の7セルあたり 2 μ L に PRO 3 の 1 の x A5 結合バッファーの 1 つの mL を含む染色液で再懸濁します。</li>
	<li>10 分間室温で暗闇の中でサンプルをインキュベートします。</li>
	<li>1 x A5 結合バッファーの 1-2 の mL を追加し、余分な汚れを削除する 6 分間 3,000 x g でサンプルを遠心分離します。 注: 混合細胞または組織サンプルは、1 x A5 結合バッファーで細胞型-特異的マーカーの組み合わせを使用して、ステップを汚す抗体を実行し、6 3,000 × g で遠心分離前に氷上 20 分 (または製造元のプロトコルに従って) 孵化させなさい分。</li>
	<li>3 mL の FACS バッファーでサンプル ペレットを再懸濁します (1 × PBS、1 x A5 結合バッファー 10 %fsc、2 ミリメートルの EDTA) 1 x 10 の7セルあたり。</li>
	<li>丸底、ポリプロピレン (フローサイトメトリー) 管に 70 μ m セル ストレーナー フィルターし、暗闇の中、氷の上のサンプルを保ちます。</li>
	<li>FACS マシンをオンにし、100 μ m ノズルを使用して標準的なセットを実行、ドロップ遅延実行、安定したストリームを確保します。</li>
	<li>サンプルをロードし、集録速度を 〜 1000 イベント/秒に設定します。</li>
	<li>FSC、SSC、APC (に-プロ-3)、FITC (A5) 電圧を調整し、集団を明確に分離することができますように FACS プロット内のイベントの位置します。</li>
	<li>20,000 イベントを記録します。</li>
	<li>パート 4 のようにゲート戦略を設定します。</li>
	<li>並べ替えレイアウト目的の並べ替えの人口として最後の ApoBD ゲートを追加します。</li>
	<li>サンプルを集録を開始し、〜 250 μ L FACS バッファーを含む新しいチューブに 5,000-10,000 の ApoBDs を集めることによってテストの並べ替えを実行します。</li>
	<li>バック フラッシュ システムを実行し、並べ替えられた ApoBDs をロードします。</li>
	<li>取得し、テストの一種 ApoBDs を記録します。</li>
	<li>ApoBD 純度が FSC (y 軸) vs A5 を比較することにより 〜 99% をあることを確認 (x 軸イベント)。 注: が A5 染色再レーザー漂白のためのサンプルを分析する際にわずかに減らすことができます。</li>
	<li>高純度を達成すると、一度元のサンプルをロードし、ApoBDs の希望の番号を取得するまでの並べ替えを続行します。 注: 必要に応じて、デュアル並べ替え実行できます ApoCells と ApoBDs を同時に分離します。 注意: ソートは長い期間にわたって、お勧め 4 ° C でコレクションの管をインキュベート</li>
	<li>並べ替えが完了したら、ApoCells、非導入と WAS アポトーシスを検証し、並べ替え後の純度を確認後並べ替え並べ替え後 ApoBDs の小さな部分を収集します。 注: テストの並べ替え、並べ替え後の純度が大きく違いはありませんが、これはストリームの設定と安定性に基づいています。</li>
</ol>3 差動遠心分離による ApoBD 分離
<ol><li>10 分間 300 x g で残りのアポトーシスのサンプルを遠心します。</li>
	<li>細胞ペレットを中断することを避けるために 〜 500 μ L を残して上清を収集し、新しい 15 mL の円錐管に追加します。</li>
	<li>残り 500 μ L を削除し、2 mL の 1x PBS (これは 'ApoCell 濃縮率' を表しますで細胞ペレットを再懸濁します。</li>
	<li>3,000 gで 20 分間収集した上清を遠心します。</li>
	<li>ペレットを確認し、(小さな細胞の小胞エクソソーム機構の解明研究などを含む上清) 上清を慎重に取り外します。</li>
	<li>1 mL の 1x PBS (これは 'ApoBD 濃縮' 分数を表します) でペレットを再懸濁します</li>
	<li>各実行可能な ApoCell 濃縮 WAS 新しい丸底、ポリスチレン (フローサイトメトリー) チューブの ApoBD 濃縮試料 100 μ L を収集します。</li>
	<li>2 x A5 結合バッファー、1: 100、A5 FITC や縮尺に PRO 3 を含む汚れの 100 μ L を追加します。</li>
	<li>暗闇の中で 10 分間室温でインキュベートします。</li>
	<li>成功したアポトーシス誘導と ApoBD 濃縮率の純度検証上記ゲートの戦略を使用して、フローサイトメトリーによるサンプルを分析します。</li>
</ol>4. フローサイトメトリー戦略をゲート
<ol><li>プロットに-プロ-3 (y 軸) に対して FSC (x 軸) すべての他の非グリセリン イベント (に-プロ-3低/中級) から壊死細胞 (に-プロ-3高) を分離します。</li>
	<li>グリセリン以外のすべてのイベントを選択し、A5 から SSC をプロットします。ゲート 1 (P1)、SSC中級/高A5低/中間細胞の人口と人口 2 (P2)、他のすべてのイベントを含む 2 つの集団。</li>
	<li>P2 に-プロ-3 A5 に対してプロットし、すべて細胞の残骸を除外する A5中級/高イベントを選択します。</li>
	<li>A5 のすべての肯定的なイベントを選択し、A5 に対して FSC をプロットします。ApoCells (FSC中級/高) から ApoBDs (FSC低) を分離します。 注: ApoBDs FACS に基づくアプローチによる ApoBD 分離のため、ゲートお勧めします最後のステップを ApoBDs に-プロ-3 A5 に比較してすべてのイベントを選択して。これにより、最終的な並べ替えゲートが FSC/SSC パラメーターではなく蛍光を使用すること。</li>
	<li>生細胞分析の P1 を選択し、ゲートの 2 つの方法のいずれかを実行します。一般生菌分析 A5 に対して FSC をプロットし、従って残りの細胞の残骸の取り外し、FSC中級/高のすべてのセルを選択します。また、詳細な分析を実行可能なセルから分離できる A5-に-プロ-3 に対して FSC をプロットすることによって初期の ApoCells。FSC中級/高実行可能なセルを選択する-プロ-3低にプロ 3中間FSC中級/高A5-初期の ApoCells。</li>
</ol>

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著者が明らかに何もありません。

1950 年代の初期、説明文からアポトーシスのフィールドは著しく、進んでいる、著名な研究領域になります。広範な努力および広範な関心にもかかわらず特定の ApoBDs の形成におけるアポトーシスの特定の側面がよく研究されていない適切な方法論の不足のため。特にアポトーシスの進行と同時に従来のフローサイトメトリー A5/PI 解析、ApoBD 分離の不純物を使用して ApoBD 形成を追跡の制限が?...

アポトーシス、プログラム細胞死のよく研究フォームは、生理的恒常性を維持し、人体1内で潜在的有害な細胞を除去する必要があります。アポトーシスの誘導後、アポトーシス細胞 (ApoCells) は一連の形態学的変化を受けるし、ApoBDs と呼ばれる小さな膜小胞に分解できます。全体的にみて、このプロセスはアポトーシス細胞分解として知られている、形態2,3に基づいて 3 段階に分けることができます。ステップ 1 (膜ブレブ) は、鉱物4,5として知られている細胞の表面に風船のような構造の形成によって特徴付けられます。ステップ 2 (アポトーシス突起形成) には、apoptopodia、apoptopodia ビーズと微小管結合スパイク6,7,8などの長い膜突起部の形成が含まれています。最後に、ステップ 3 (ApoBD 編成) にはアポトーシス突起および/または ApoBDs6,9を生成する ApoCells の断片化が含まれています。前の調査結果は、アポトーシス細胞のクリアランスを支援し、細胞間のコミュニケーションを仲介することで ApoBDs の役割を示唆しています。たとえば、ApoBDs に、ApoCell の断片化が小さい '' 作品を一口食2,10,11を囲むことによって簡単に削除することができますを生成可能性があります提案します。さらに、ApoBDs は、DNA、RNA および蛋白質は、細胞間コミュニケーション12,13,14を容易にする周囲の細胞に取り引きされる可能性がありますなどの生体分子のシリーズを抱く可能性があります。これらのプロセスを調べる機能的、アポトーシス誘導および ApoBD の形成の検証 (i)、ApoBDs の分離 (ii) と (iii) ApoBD 純度の確認を含む 3 つの重要なパラメーターを確認する重要です。
以前は、アポトーシスと ApoBDs15,16,17,18の勉強をフローサイトメトリーと電子顕微鏡を含むメソッドの数が使用されています。しかし、ApoBD の検出と定量、しばしば困難または見落とさ。例えば、最も日常的に使用される流れのフローサイトメトリーによるアポトーシス アッセイ アネキシン V を採用 (A5、外部結合タンパク質 'を食べる-私' 信号ホスファチジルセリン (PtdSer)) 核酸染色 propidium ヨウ化 (PI)19。しかし、この普遍的な染色の組み合わせを使用して、解析では、のみ 3 種類のサンプルで (実行可能なセル、ApoCells と壊死細胞) 細胞のサブセットがあること想定しています。さらに、cytometry 流れの試金の apoptosis のため多くの研究者によって「ゴールド スタンダード」として考慮される、しかし、以降のデータ解析はしばしば FSC/SSC中間高イベントの選択最初のゲーティング ステップを通じて ApoBDs を除外します。したがって、A5 とする PRO 3 を使用して新規流れ cytometry 試金を最近開発した、7、20チャンネルは別の核酸染色カスパーゼ 3/7 活性化パネキシン 1 (PANX1) によって選択的に取ることができます。カスパーゼ 3 による PANX1 活性化はアポトーシスの早い段階での PtdSer 露出を前に、ようにプロ 3 は特異的アポトーシスとネクローシス細胞を汚れ。このアプローチ戦略をゲートの小説と組み合わせてさらに、データ分析中に取得しているすべてのイベントが含まれています、6 セル/パーティクルのサブセットの識別結果としてを含む: (i) 実行可能なセル (FSC/SSC中級/高、A5低にプロ 3低)、(ii) A5-初期の ApoCells (FSC/SSC中級/高A5低、中間にプロ 3)、(iii) A5+ ApoCells (FSC/SSC中級/高、A5高にプロ 3 の中間)、(iv) 壊死細胞または後期 ApoCells (FSC/SSC中級/高A5高にプロ 3高)、(v) ApoBDs (FSC/SSC低、A5中間に-プロ-3低中間/)、および (vi) 破片 (FSC/SSC低A5低、低にプロ 3)20。我々 のアプローチでは、すべてのセル/パーティクルのサブセットと、もっと重要なは、細胞および破片20からの ApoBDs の分離分析の重要性を強調しています。したがって、このアプローチは、アポトーシスおよび ApoBD の形成の誘導を同時に検証する効率的な方法を示します。
伝統的に、ApoBDs は、さまざまな細胞や密度に基づく他の細胞小胞から ApoBDs を分離できるという差動遠心分離アプローチを通じて分離されています。しかし、このようなの遠心分離法はしばしば低 ApoBD 純度、定量ステップ サンプル純度および/または細胞型特異 ApoBDs17,21,22を分離することができないことを確認するための不足によって制限されます。そこで、最近 2 つのアプローチ、FACS に基づいた新しい差動遠心分離に基づくアプローチをアポトーシスとサンプルの純度のための23の誘導を検証する私たち以前に確立された流れフローサイトメトリー法と結合することができますを開発しました。FACS 手法による ApoBD 分離を最大 99% 純度 ApoBDs を豊かにすることができます、さまざまな混合細胞集団、組織サンプルと体液23から ApoBDs を分離する細胞型特異抗体と組み合わせて利用できます。さらに、我々 の改訂された差動遠心分離アプローチを示しますに ApoBDs を分離するための効率的な方法 > 90% 純度23。
本稿で我々 はアポトーシス誘導を検証し検出し、定量化の ApoBD 形成私たちの実験の詳細について説明します。FACS ベースおよび差動遠心分離ベースのメソッドを使用して ApoBD 分離ワークフローはまた詳述し、比較しました。代表的なデータ記述の方法論では、将来の ApoBD 研究の効果的な最先端のツールをデモンストレーションします。

<table><tbody><tr><td>Cells e.g. cultured human THP-1 monocytes (clone number: TIB-202)</td><td>ATCC</td><td>-</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Life Technologies</td><td>22400-089</td><td></td></tr><tr><td>Penicillin-streptomycin mixture</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>FSC</td><td>Gibco</td><td>10099-141</td><td></td></tr><tr><td>1x PBS</td><td>-</td><td>-</td><td></td></tr><tr><td>Annexin V FITC</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>TO-PRO-3 iodide</td><td>Life Technologies</td><td>T3605</td><td>TO-PRO-3 may cause skin, eye and respiratory irration. Avoid direct contact.</td></tr><tr><td>10x Annexin V binding buffer</td><td>BD Bioscience</td><td>556454</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>1001710526</td><td></td></tr><tr><td>Centrifuge tube (15 mL)</td><td>Cellstar</td><td>188271</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 mL)</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>Tissue culture incubator (37 &amp;deg;C, 5% CO2)</td><td>-</td><td>-</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>392932</td><td></td></tr><tr><td>FACS ARIA III Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Canto II Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Diva 6.1.1 software</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FlowJo 8.8.6 software</td><td>-</td><td>-</td><td></td></tr><tr><td>&amp;nbsp;UV Stratalinker 1800</td><td>Stratagene</td><td>-</td><td></td></tr></tbody></table>

detection and isolation of apoptotic bodies to high purity

アポトーシス小体 (ApoBDs) 機構の解明、エクソソーム細胞小胞族最大の型の一つである ApoBDs の主要メンバーであります。ApoBDs が細胞除去として人身売買生体分子を介して細胞間のコミュニケーションを助けることができることが提案されています。使用の識別と ApoBDs の分離従来多くの場合、正確な定量化と低いサンプル純度の欠如によって制限されます。ここでは、アポトーシスの誘導の確認、ApoBD 形成を検証および ApoBDs を高純度に分離するワークフローについて述べる。またアウトライン、蛍光活性化セル (FACS) を並べ替えと ApoBDs を分離に基づく差動遠心分離アプローチを比較します。使用して孤立した ApoBDs の純度を確認、さらに、以前の流れ cytometry による染色と分析法を確立します。まとめると、記述されているアプローチを使用して THP 1 単球のアポトーシスとアポトーシス細胞の分解は誘発され、検証と THP 1 球から生成された ApoBD は 97-99% の純度に分離されました。

ここで説明する手順を使用して、THP 1 単球のアポトーシスが誘導されたと ApoBDs が検出および FACS ベースまたは差動遠心分離アプローチ (図 1) のどちらか経由で分離されました。まず、紫外線照射によるアポトーシス誘導し、細胞アポトーシスの形態、ブレブ、アポトーシス膜突出部形成 ApoBDs6の生成などを展示したとき 2-3 時?...

Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

Detection and Isolation of Apoptotic Bodies to High Purity

検出、定量化およびアポトーシス サンプルから高純度にアポトーシス小体を分離する流れの cytometry または差動遠心分離を使用してワークフローを開発しました。

高純度にアポトーシス小体の検出と分離

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

detection-and-isolation-of-apoptotic-bodies-to-high-purity

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

10.5K ビュー.  La Trobe University. 検出、定量化およびアポトーシス サンプルから高純度にアポトーシス小体を分離する流れの cytometry または差動遠心分離を使用してワークフローを開発しました。

ビデオ: Detection and Isolation of Apoptotic Bodies to High Purity

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

アポトーシス反転後新しく形成された乳癌幹細胞様細胞の流れフローサイトメトリー法による検出

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

がん研究

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

監視劈開カスパーゼ-3活性およびカリウム誘発性膜の脱分極に続いてライブセルイメージングとCleaveable蛍光色素基質を用いて不死化オリゴデンドログリア細胞のアポトーシス

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

神経科学

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

アクチノマイシンD処理SiHa子宮頸癌細胞におけるアポトーシスバイオマーカーのフローサイトメトリー解析

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

視床下部細胞モデルでアポトーシスを決定するために、カスパーゼの多重化アッセイの使用

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

蛍光アッセイまたはフローサイトメトリーを使用したカスパーゼ活性の測定

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

生化学

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 ...

生物学

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

単一細胞の蛍光顕微鏡による Efferocytosis の定量化

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

免疫・感染学

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

マクロファージに貪食アポトーシス胸腺細胞の実験的解析

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

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 ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

隣接セル受けてアポトーシス細胞死との相互作用によって生細胞に誘導されるイベントを細胞内シグナルの同定

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...