この作品は、ローマ ・ ラ ・ サピエンツァ大学とイタリアの嚢胞性線維症研究財団 (プロジェクト FFC #11/2014 年シエナ、ソンドリオ Valchiavenna、Cerea Il ソリーゾ ・ ディ ・ ジェニー、パヴィア FFC 代表団によって採用) からの資金によって支えられました。この仕事の一部はまたによって FILAS グラント Prot. FILAS-RU-2014-1020 をサポートされていました。
気管支の上皮細胞を提供するため博士ロレッタ フェレーラ (U.O.C. Genetica ・ メディカ、Istituto Giannina ガスリーニ、ジェノバ、イタリア) に感謝しております。

1. セル準備
<ol><li>10% 牛胎児血清 (FBS)、抗生物質 (ペニシリンとストレプトマイシンの 0.1 mg/mL) およびピューロマイシン プラス 2.5 x 106 10 mL の最小必須培地 (MEM) 2 mM グルタミン (MEMg) を添加した細胞を播く (0.5 μ G/ml の選択と維持のため、行のセル) T75 フラスコ。2 日間の 37 ° C、5% CO2でフラスコを孵化させなさい。実験を始める前に、倒立顕微鏡下で細胞の合流点を確認します。 注: 実験用セルは、レンチウイルスベクターをピューロマイシン耐性で導入した不死化ひと気管支上皮細胞です。安定機能 CFTR14,15を表します。</li>
	<li>セルの合流は 90-100% に達したら、フラスコから培地を吸引し、生物学的安全キャビネット クラス II の下で廃棄ボトルにそれを破棄します。カルシウムとマグネシウム (CMF PBS) なし生理食塩水リン酸緩衝の ml の 6 セルを洗います。優しく、フラスコを手動でロックし、CMF PBS を破棄します。 注: ピペットで細胞膜を触れないように気をつけてください。</li>
	<li>CMF PBS の 10 mL を追加し、10 分間の 37 ° C および 5% の CO2でフラスコを孵化させなさい。</li>
	<li>CMF PBS を吸引し、それを破棄します。トリプシン/EDTA の 2 mL をフラスコに追加します。</li>
	<li>完全に細胞が顕微鏡の下で目に見えて戸建まで 10 分の CO2を 37 ° C、5% でフラスコをインキュベート細胞をコートしソリューションを許可するフラスコみ合います。 注: インキュベーション時間の終わりに、セルが丸いとプラスチック表面にもアタッチされていないに表示されます。まあ戸ないセルは、手動撹拌が必要かもしれません。</li>
	<li>MEMg プラス 10% の 10 mL を加えてトリプシンを不活化し、フラスコの底を洗浄することにより細胞を収集する政府短期証券。50 mL のコニカル チューブにボリュームを転送します。</li>
	<li>80 x gで 5 分間チューブを遠心します。</li>
	<li>上清を吸引し、再 MEMg プラス 10% の 6 mL のセルを中断政府短期証券。ピペット繰り返し任意の塊を破る。</li>
	<li>マイクロ ピペットで細胞を懸濁液の 10 μ L を取り出して、以前 Burker またはノイバウアー室の上に置くカバー ガラスの下でボリュームを挿入します。</li>
	<li>セル数をカウントします。</li>
</ol>2. 細胞文化の播種を挿入します。
<ol><li>12 ウェル プレートの各ウェル、滅菌ピンセット (図 1) で文化挿入を転送します。板の表面にそれらを修正するために挿入のエッジに沿って押すようにピンセットを使用します。 注: 挿入により接着性粘着性の下側があります。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56825/56825fig1.jpg"> 図 1: 12 ウェル プレートのウェルに正しく入れて、シリコーン文化挿入の略図。<a href="//cloudflare.jove.com/files/ftp_upload/56825/56825fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol start="2"><li>正しく MEMg プラス 10% の細胞懸濁液を希釈 FBS。細胞懸濁液 (約 3.5x104セル/室) の 70 μ L で、挿入の各区画を入力します。 注: 各区画に適用されるセルの密度は、細胞の種類によって異なります。24 h 内で合流の細胞密度を使用することをお勧めします。</li>
	<li>顕微鏡で細胞挿入コンパートメントから漏れていないと 37 ° C、5% CO2で 24 時間 12 ウェル プレートを孵化させなさいことを確認します。</li>
</ol>3. 擬似創傷治癒アッセイ
<ol><li>インキュベーション後、コンフルエントの細胞が形成されていることを確認を倒立顕微鏡下で細胞を可視化します。</li>
	<li>再 1 ml MEMg のテスト混合物 (例えばアンプ) を中断します。 注: 準備新鮮なアンプ希釈原液-20 ° C で保存から始まって</li>
	<li>滅菌ピンセットでチップを慎重に取り外します。細胞膜を壊さないように注意してください。転送吸収紙の上に挿入します。 注: 同じ挿入を再利用するには、滅菌に少なくとも 3 時間の 70% エタノールで。その後にそれらを捨てることをお勧めします。</li>
	<li>非付着性のセルを削除するには、マイクロ ピペットを使用してあたり MEMg の 1 mL を追加します。プレートを閉じ、そっとそれをロックします。 注: は、混乱を避けるために細胞膜上に直接メディアを追加しないでください。</li>
	<li>培地を吸引し、ウェルあたり MEMg の 1 mL と置き換えます。プレートを閉じ、ビデオ カメラを装備して、4 倍の倍率で倒立顕微鏡下で (挿入によって作成された) 携帯無料ギャップを可視化します。画像時 0 (T0) を取得し、.jpg 形式で保存します。</li>
	<li>井戸からメディアを取り出して、1 mL の PBS で洗浄し、それを破棄します。</li>
	<li>井戸に (3.2 の時点で準備) テスト混合物を追加します。無処理試料 MEMg の 1 mL を追加します。37 ° C と 5% の CO2でプレートを孵化させなさい。</li>
	<li>15、20、および 24 時間治療後、4 倍の倍率の顕微鏡下で細胞の移動を観察し、画像を取得します。 注: この手順では、T0 は同じエリアの画像をキャプチャする試み。イメージがキャプチャされます時間間隔は、セルの移動速度によって決まります。</li>
	<li>いくつかの選択的阻害薬の効果を研究、すなわちAG1478、細胞移動への挿入を削除する前に各挿入のコンパートメントから媒体を吸引します。<ol>
<li>新鮮な MEMg と各コンパートメントを洗浄し、AG1478 を添加した MEMg の 70 μ L でそれを埋めます。</li>
		<li>37 ° C および 5% CO2で潜伏の 30 分後 3.3 ポイントから説明するように進みます。 注意: 洗浄のステップおよび細胞特異的阻害剤単分子膜の前処理中に挿入を削除しないように注意します。</li>
	</ol>
</li>
</ol>4. 画像解析
<ol><li>実験が完了したら、さまざまな実験的グループの最も代表的なサンプルの画像を選択し、T0、T15、T20、T24 h で 1 つの画像を含む zip ファイルを作成します。 注: 単一の画像はすべてサンプルを選択した時間間隔で撮影されます。少なくとも 3 回を繰り返し、それぞれの実験の実行をトリプリケートします。最後に、すべての実験的グループ、("a"、"b"、"c"、等、それぞれの独立した実験から派生する) 3 つの画像の最小の各時点で分析されます。</li>
	<li>自動的に電子メールで選択した時点で (パーセンテージ) 細胞に覆われたとスクラッチ領域の実験的データを含むスプレッドシートの概要ファイルを提供する画像解析ソフトに zip ファイルをアップロードします。 注: リーディング エッジとギャップ領域の認識は (目的で画像の明るさが大幅に変更された点を識別する) エッジ検出法に基づいて。</li>
	<li>データを保存し、それらを収集します。時間 0 で平均値に対するすべてのデータを正規化します。各時点で相対的標準誤差 (SEM) すべて複製の正規化されたデータの平均値を計算します。双方向分散分析 (ANOVA) を使用して、適切な統計解析ソフトウェアの統計分析を実行します。ペプチド投与群と異なる時間間隔で制御グループの違いと見なされる統計的に重要なpの < 0.05。</li>
	<li>グラフに、選択した時間間隔すべてサンプル グループ対の細胞で覆われた領域のパーセントを示すヒストグラムとして得られたデータをプロットします。</li>
</ol>

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著者がある何も開示するには

細胞遊走は、創傷治癒、萌芽期の開発、がんの転移など多くの生理学的および病理学的イベントに欠かせないプロセスです。細胞移行生体外で勉強する基本的な手順を含む: (iii) 傷までのさまざまな時間間隔での画像キャプチャ閉鎖に達する (i) 細胞膜、(ii) 細胞の合流の層で擬似傷の生産の作成、および (iv) 選択したセルの移行速度を定量化するために, 動画像の解析。
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動物の創傷治癒過程がけが1後の整合性と組織層の正常な機能に再確立する基本的なプロセスであることを主として知られています。物理・化学的侮辱から防護壁を形成上皮表面 (例えば、呼吸器、皮膚、消化管) の外部環境にさらされているにもかかわらず、傷の形成が発生しやすく、特に後手術または微生物感染症2。例として、嚢胞性線維症 (CF) 患者において日和見細菌菌緑膿菌、によって肺組織の植民地化とそれに伴う呼吸不全3、気道上皮の損傷につながる 4。創傷治癒は、負傷した組織5通常の建築を復元する複雑なホスト修理メカニズムです。上皮形成、血管新生、コラーゲン産生と細胞分化6,7,8 組織の改造現象を包括的な再生期間続いて初期の炎症によって特徴づけられる.上皮の完全性を確保するため、微生物の増殖を制御するため、すべての生きている有機体は抗菌ペプチド (アンペア)9,10を含む防衛分子を生成します。創傷治癒過程シミュレートの in vitro細胞残屑および異なる種類の細胞間の複雑な相互作用の欠乏のために非常に困難です。ただし、ペプチドの生体外で能力を刺激することによって擬似傷の閉鎖を加速する上皮細胞の移行は侵害された上皮を治癒する能力を示す。確かに、細胞遊走は創傷治癒にレート制限イベントと改良された創傷治癒のためのターゲット治療に役立つ細胞の移動に影響を与える要因を勉強します。
ここでは、再現性の高い実験的試金はセル移行体外を評価する特殊シリコン文化挿入に基づいて提供しています。それは、コンフルエントの細胞膜上に 500 μ m ギャップ (擬似傷) の作成に基づいています。無料のセル領域に移行する、新しい細胞間の接触を形成、人工の「負傷者」のフィールドの端にあるセルが開始されます。文化の挿入は、高速治癒促進実験のための新しいツールを表します。500 μ m の壁で区切られた 2 つの貯水池が提供され、12 ウェル プレートの井戸、3 cm 皿プレートにそれら正しく配置ことができます。挿入の各コンパートメントの細胞懸濁液を充填セル挿入の除去は (分離壁と同じ幅) 約 500 μ m のきれいな無細胞のギャップを生む一方、confluence (コンフルエンス) まで各指定地域で成長することができます。試験化合物を添加した適切な細胞培養液を皿に追加できるプレート/ウェル。その後、ギャップの閉鎖は、好ましくは 1 つの画像取り込みビデオ カメラ搭載、倒立顕微鏡下で異なる時間間隔で視覚化できます。最後に、web ベースの自動画像解析プログラムによってセル面積の変化の測定には、創傷閉鎖と細胞移動の速度の定量化ができます。全体的にみて、この方法は、スクラッチは滅菌した針でコンフルエントの細胞膜を切開によってなされる手動でまたはピペット チップ11クラシックのアッセイに関して一歩前進です。確かに、最後の手順は、皿のプラスチック底を破壊できるプレート/まあ、表面コーティング、しわを作成します。さらに、高針の先端に研究者によって適用される圧力にもよりますので、「負傷者」の領域には、ギャップの全体の長さに沿って明確に定義された幅がありません。さらに、剥がれ細胞が、ゼロの端の生活および死んだ細胞の塊を形成できます。また、「負傷者」の領域に生きている細胞の広がりは、12非再現性のある結果を生成する細胞移動の速度と干渉することができます。
さらに、スクラッチのイメージ解析プラットフォームのおかげでユーザー急速に受信できる (数分) 追加のソフトウェアを取得する必要なく選択したセルの回遊行動の定量的なデータ。このプラットフォームは、低 (~ 5 X) の位相コントラスト顕微鏡画像、中規模 (~ 10 X)、および (~ 20 倍) の高倍率を分析が可能です。セルの速度だけでなく、細胞に覆われたエリアとスクラッチ領域の割合を示すサマリー ファイルを生成する (*.jpg、*.jpeg、*.jp2, *.png, *.gif, *.tiff 形式) で画像の zip ファイルをアップロードした後、分析は自動的に行なわれます移行は、異なる時間間隔で。
カエル皮膚のアンプ誘導体、すなわちEsc(1-21) とそのジアステレオマー Esc(1-21)-1 c13、および機能 CF 膜コンダクタンス レギュレータ (CFTR)14,15を表現する気管支細胞ラインを使用して、この作業でペプチドによる細胞移行 (無処理) サンプルとの比較の例を示します。気道上皮、CFTR が肺機能を維持するために重要な役割を果たすことに注意してください、修理16を傷します。さらに、による上皮成長因子受容体 (EGFR)、前述のペプチドによる気管支の細胞の移動を含む EGFR12の活性化の証拠の選択的阻害剤 (例えばAG1478)17 18の報告します。
この手順の目標は文化そのようなシリコンを挿入の使用状況が正確に付着性のセル (例えば、気管支上皮細胞) と分子の移行を判断する迅速かつ簡単にアクセスできるアッセイをどのように表現するかを要約すると、このようなイベントを制御するメカニズムです。

<table border="0" cellpadding="0" cellspacing="0" width="852">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Minimal essential medium (MEM)&#160;</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB2071L</td>
			<td style="width:256px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Glutamine</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heat inactivated Fetal Bovine Serum (FBS)</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECS0180DH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin and Streptomycin</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3001D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Puromycin</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypsin/EDTA 1X in PBS</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3052D</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">DPBS without calcium and magnesium (CMF-PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DPBS with calcium and magnesium (PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ibidi Culture-Insert 2 well</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">80209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Wimasis Image Analysis</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">30002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PRISM software&#160;</td>
			<td>GraphPad</td>
			<td style="width:119px;">version 6.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vitro wound healing assay to evaluate cell migration

この作業の目的は、抗菌ペプチド (アンペア)、細胞の移動を刺激するなどのいくつかの免疫調節分子の能力を評価する新しい方法を提示することです。重要なは、細胞遊走、傷害後の整合性と組織層の正常な機能に再確立する創傷治癒プロセスの間にレート制限イベントです。手動で作られた単層細胞傷に基づいている古典的な分析上のこのメソッドの利点は、特殊シリコン文化の使用は明確に定義された幅 (500 μ m と無細胞の擬似巻きフィールドを作成する 2 つのコンパートメントを提供するを挿入します).さらに、自動画像解析プラットフォーム、ため急速に創傷閉鎖と細胞移動の速度で定量的なデータを得ることが可能です。正確には、気管支上皮細胞の移動に 2 つのカエルの皮アンプの効果が表示されます。さらに、これらの細胞特異的阻害剤での前処理は分子機構などのイベントに関する情報を提供します。

このプロトコルは創傷治癒機能 CFTR を表現する気管支上皮細胞に誘発される細胞の移行活動の面で Esc(1-21) と Esc(1-21)-1 c の効果を決定するために使用されました。このアッセイで文化挿入された 12 ウェル プレートの井戸に置かれ、各コンパートメントは 35,000 MEMg 10% を添加した細胞が播種された FBS。セルその後 24 時間以内の完全な合流点に達した、500 μ m ギャップ?...

Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

ここでは、ペプチドの気管支上皮細胞の移動に及ぼす影響を評価するためのプロトコルを提案する.このメソッドは、セル移行および創傷閉鎖の速度に定量的なデータの迅速かつ再現性の高い国又できます。

新規In Vitro細胞移動を評価する癒しのアッセイを傷

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

a-novel-in-vitro-wound-healing-assay-to-evaluate-cell-migration

Research

JoVE Journal

Immunology and Infection

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

21.6K ビュー.  Sapienza University of Rome. ここでは、ペプチドの気管支上皮細胞の移動に及ぼす影響を評価するためのプロトコルを提案する.このメソッドは、セル移行および創傷閉鎖の速度に定量的なデータの迅速かつ再現性の高い国又できます。

ビデオ: A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白血球ホーミングと移行の非侵襲的イメージング in vivoで</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

免疫・感染学

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

腸管指向性T細胞の移動を研究するために競争力のあるホーミングアッセイ

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokineticトラック運動アッセイによる細胞遊走の定量的評価

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

インビトロアッセイで HIV特異的CD4 T細胞エフェクター機能に対する免疫調節経路の影響を評価する

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

インビトロ 病原体誘導性好中球トランス上皮移動を評価するコーカルチャーアッセイ

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

顆粒球機能の変化を評価するために、イメージベースのフローサイトメトリー技術<em&gt;インビトロ</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

での感染の間に流入領域リンパ節へのマウスの皮膚樹状細胞の移動を研究するためにCFSEベースアッセイ<em&gt;マイコバクテリウム・ボビス</em&gt;カルメット - ゲラン

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

ヒト好中球由来の巨大食細胞の新規亜集団の開発および同定<em&gt;インビトロ</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Fcγ受容体媒介食作用を評価するために、単球単層アッセイの使用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

生体外でマウスのナチュラル キラー細胞の大量生産のため新規無料の送り装置システム

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...