Name: flow cytometry-based assay for the monitoring of nk cell functions
Uploaded: 2016-10-30T15:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry-based Assay for the Monitoring of NK Cell Functions at JoVE.com

Authors were supported by the German Cancer Aid (Max Eder Nachwuchsgruppe, Deutsche Krebshilfe; EU), the LOEWE Center for Cell and Gene Therapy Frankfurt (EU, ST) funded by the Hessian Ministry of Higher Education, Research and the Arts, Germany (III L 4- 518/17.004) and by the "Alfred- und Angelika Gutermuth-Stiftung", Frankfurt, Germany (EU). BJ was funded by a Mildred Scheel postdoctoral scholarship from the Dr. Mildred Scheel Foundation for Cancer Research. ST was founded by a GO-IN postdoctoral fellowship (PCOFUND-GA-2011-291776). The authors thank Becton Dickinson (BD) for providing the FACSCanto II and Canto10c Flow Cytometry Analyzers used in this study.

この研究は、フランクフルト大学の地域倫理委員会の勧告に準じて行きました。 K562細胞の1培養<ol><li> R10培地で培養K562細胞を37℃および5％CO 2で細胞培養フラスコ中で1ml当たり0.5×10 6細胞の密度で（グルタミン培地、1％ペニシリン/ストレプトマイシン、10％ウシ胎児血清を有するRPMI1640） 2。 </li><li>収穫K562細胞の新しい実験の開始前に24時間。 <ol><li>インキュベーター...

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記載された方法は、健康なドナーまたは患者の全血試料からのNK細胞の機能を研究するための、簡単で迅速かつ信頼性の高い方法です。この方法は、他の多くの精製方法15のために必須である時間のかかる密度勾配遠心分離を避け、直接全血からNK細胞を精製するための大きな利点を提供しています。また、それは、小児及び/又は免疫不全の患者のサンプルのために適切な代替になり?...

先天性免疫系ナチュラルキラー（NK）の一部として、細胞はウイルス感染や悪性形質転換した細胞に対する防御の第一線に寄与する。抑制と活性化受容体のシステムは、T細胞とは対照的に、前の抗原プライミングすることなく、健康で形質転換された細胞を区別できるようにします。標的細胞に遭遇すると、NK細胞は、それらの標的を殺すために免疫シナプスに自分の細胞傷害性顆粒（ 例えば、パーフォリン、グランザイム）の内容物を放出します。また、NK細胞が生産し、異なるサイトカインの種類（ 例えば、インターフェロンγ：;：TNF-α、IFN-γ、腫瘍壊死因子α）を分泌：標的細胞上に及びケモカイン（MIP-1β 例えば、マクロファージ炎症性タンパク質1βを）相互作用またはサイトカイン刺激1。 このような細胞毒性、ケモカインおよびサイトカイン産生として十分なNK細胞の機能は、多様なDISの運命に重要な影響を持っています容易になります。それらは減少IFN-γ産生およびNK細胞受容体2の活性化の低下した発現からなる診断で不良NK細胞プロフィールを示す場合、白血病患者は、再発率の増加を示します。標的細胞の相互作用の際にサイトカイン産生などNK細胞の数および機能の早期回復は、同種幹細胞移植3を受けている患者において減少再発および生存率の改善率と関連しています。また、C型肝炎ウイルスに感染した患者におけるインターフェロン療法の開始時に、末梢NK細胞の脱顆粒の容量は、非応答者4よりも早い応答における強いです。リンパ腫または多発性骨髄腫を患っている患者における自家幹細胞移植（autoSCT）後15日目のNK細胞数（&gt; 80 /μl）を改善された無増悪および全生存5を予測しています。メラノーマ患者におけるT細胞免疫グロブリンの発現およびムチンドメインcontaininG分子3（TIM-3）、NK細胞上の免疫調節タンパク質は、病期および予後6と相関します。 科学者たちは、過去数十年間を通じてNK細胞の機能を監視しています。事前のプライミングなしの腫瘍細胞に対するNK細胞の細胞毒性の最初の分析は、51 Cr放出アッセイ7を使用して対処しました。より最近では、科学者は、拡張NK細胞8の細胞毒性を評価するために、非放射性の方法を開発しました。サイトカインおよびケモカイン産生は、しばしば、酵素結合免疫吸着アッセイ（ELISA）技術9,10を用いて評価されています。過去数十年の間、これらの方法は、フローサイトメトリーに基づくアッセイによって補完されています。従来の表面染色プロトコルとの組み合わせでタンパク質輸送阻害剤（ 例えば、ブレフェルジンAおよびモネンシン）と細胞透過法の使用は、異なる特定のリンパにおけるケモカインおよびサイトカイン産生を研究する科学者を有効にしていますocyteサブセット（ 例えば 、T、BまたはNK細胞）11。また、別のフローサイトメトリーベースのアッセイは、T及びNK細胞の細胞毒性をモニターするために開発されてきました。 2004年アルターらは 、細胞傷害性顆粒12の脱顆粒のマーカーとして標的細胞の遭遇時にNK細胞のリソソーム関連タンパク質のCD107a（LAMP1）の表面発現を記載しました。異なる蛍光色素及びマルチチャネルフローサイトメーターの広い範囲は、私たちの日に利用可能であるので、同時に異なるNK細胞サブセットに多様なNK細胞機能（細胞毒性、サイトカインおよびケモカイン産生）を監視することが可能となりました。これは、生検または白血球減少症を患っている患者の血液サンプル中、 例えば、サンプルサイズが制限される状況において特に重要となります。 グローバルNK細胞の機能をテストするために、別のフローサイトメトリーに基づくアッセイを効率的に組み合わせることができます。テオレルらは HEAからNK細胞を刺激し腫瘍細胞株K562とlthyドナーと13、フローサイトメトリーを介して、NK細胞の脱顆粒、インサイドアウト信号およびケモカイン産生を分析しました。最近autoSCT中の腫瘍患者におけるNK細胞のサブグループ、表現型および機能はフローサイトメトリーに基づくアッセイを流す用いて分析しました。これは、NK細胞はautoSCT 11後の非常に早い時点での腫瘍細胞の認識の際に、サイトカイン/ケモカインの脱顆粒及び生成することができたことが示されました。 ここでプロトコルが同時に異なるサブセット内のNK細胞の機能を監視することを可能にするフローサイトメトリーに基づくアッセイを使用して脱顆粒の容量、ケモカインおよびサイトカイン産生を含む、腫瘍細胞との相互作用にNK細胞の機能を評価するために記載されています。

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 + glutamine</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">6187-044</td>
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			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
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			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">10270-106</td>
			<td style="width:167px;">heat inactivated before use</td>
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			<td height="42" style="height:42px;width:216px;">ammonium-chloride-potassium (ACK) lysis buffer</td>
			<td style="width:135px;">made in house</td>
			<td style="width:119px;">/</td>
			<td style="width:167px;">components are listed in the text</td>
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			<td style="width:135px;">Fresenius Kabi</td>
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			<td>Life Technologies</td>
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			<td>Lonza Group Ltd</td>
			<td style="width:119px;">BE04-743Q</td>
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			<td style="width:135px;">DRK Blutspendedienst, Frankfurt/M&#160;</td>
			<td style="width:119px;">/</td>
			<td style="width:167px;">healthy donors with blood type AB; heat inactivated before use</td>
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			<td style="width:135px;">Marienfeld superior/ LO-Laboroptik Ltd.</td>
			<td style="width:119px;">0640030/ 1110000</td>
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			<td height="21" style="height:21px;width:216px;">trypan blue solution (0,4%)</td>
			<td>Invitrogen</td>
			<td style="width:119px;">15250-061</td>
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			<td style="width:135px;">Corning&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Falcon 96 Well Round Bottom Not Treated Microplates</td>
			<td style="width:135px;">Corning&#160;</td>
			<td style="width:119px;">351177</td>
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			<td height="25" style="height:25px;width:216px;">DPBS (Ca2+- and Mg2+-free)</td>
			<td style="width:135px;">Gibco Invitrogen</td>
			<td style="width:119px;">14190-169</td>
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			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">A2153-100G</td>
			<td></td>
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			<td height="25" style="height:25px;width:216px;">NaN3</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">08591-1ML-F</td>
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			<td height="42" style="height:42px;width:216px;">phorbol 12-myristate 13-acetate (PMA)&#160;</td>
			<td style="width:135px;">Merck</td>
			<td style="width:119px;">524400-1MG</td>
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			<td height="42" style="height:42px;width:216px;">ionomycin&#160;</td>
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			<td height="21" style="height:21px;width:216px;">interleukin 15 (IL-15)</td>
			<td>PeproTech</td>
			<td>200-15</td>
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			<td height="21" style="height:21px;width:216px;">Proleukin S (IL-2)&#160;</td>
			<td style="width:135px;">Novartis Pharma</td>
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			<td height="126" style="height:126px;width:216px;">Golgi Stop, Protein Transport Inhibitor (containing Monensin)</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:119px;">554724</td>
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			<td height="42" style="height:42px;width:216px;">Golgi Plug, Protein Transport Inhibitor (containing Brefeldin A)</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:119px;">555029</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:135px;">AppliChem</td>
			<td style="width:119px;">UN2209</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">saponin</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">47036</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">flow cytometer: Canto10C</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:119px;">/</td>
			<td></td>
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			<td style="width:135px;">TreeStar Inc.</td>
			<td style="width:119px;">/</td>
			<td></td>
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			<td style="width:135px;">Graph Pad Inc.</td>
			<td style="width:119px;">/</td>
			<td></td>
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			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-098-308</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">MACSxpress NK isolation kit</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-098-185</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">MACSxpress Erythrocyte Depletion Kit, human</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-098-196</td>
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			<td style="width:119px;">130-090-753</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">anti-human CD3 APC</td>
			<td style="width:135px;">Biolegend</td>
			<td>300412</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">anti-human CD3 V450</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>560366</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">anti-human CD14 PerCP</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td>130-094-969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD14 V450</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>560349</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD16 PE</td>
			<td style="width:135px;">Biolegend</td>
			<td>302008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD16 PerCP</td>
			<td style="width:135px;">Biolegend</td>
			<td>302029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD19 PE-Cy7</td>
			<td style="width:135px;">Biolegend</td>
			<td>302216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD19 V450</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>560353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD45 BV510</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>563204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD56 FITC</td>
			<td style="width:135px;">Biolegend</td>
			<td>345811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD107a PE</td>
			<td style="width:135px;">Biolegend</td>
			<td>328608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human IFN-&#947; AF-647</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>557729</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human MIP-1&#946; APC-H7</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>561280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:135px;">Biolegend</td>
			<td>422801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zombie Violet Fixable Viability Kit</td>
			<td>Biolegend</td>
			<td>423113</td>
			<td>fixable dead cell marker</td>
		</tr>
	</tbody>
</table>

flow cytometry-based assay for the monitoring of nk cell functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

<html>全NK細胞集団と三つの異なるNK細胞サブセットの脱顆粒、サイトカインおよびケモカイン産生を分析するためのゲーティング戦略は、図1に示されています。 1健康なドナーの代表的な結果を図2に示されている。任意の刺激のないNK細胞はいずれもIFN-γもMIP-1βを生産し、その表面上のCD107a（ <str...

Watch this Scientific Journal Video about Flow Cytometry-based Assay for the Monitoring of NK Cell Functions at JoVE.com

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

簡単かつ信頼性のある方法は、異なるNK細胞サブセット内の脱顆粒、サイトカインおよびケモカインの産生などNK細胞機能のセットを分析するために、ここで説明されています。

NK細胞の機能を監視するためのサイトメトリーに基づくアッセイを流します

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Research

JoVE Journal

Immunology and Infection

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

使用して樹状細胞の移行の監視<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁気共鳴イメージング

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

「マスト細胞ノックイン」マウスを用いた生体内肥満細胞の機能解析

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

虚血性マウス脳からの免疫細胞の単離およびフローサイトメトリー分析

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

マウスの骨髄から形質細胞様樹状細胞の精製のために、蛍光活性化細胞選別

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

網膜ミクログリアの貪食機能を評価します<em&gt;インビボ</em&gt;フローサイトメトリーベースのアッセイを使用しました

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

ヒト NK 細胞活性の評価の流れのフローサイトメトリーを用いた細胞毒性の試金

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

単純なフローサイトメトリーによるジカ回復期血清を用いたデング ウイルスのIn Vitro抗体依存強化測定法

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

ミトコンドリア、リソソーム T 細胞のマルチカラー フロー フローサイトメトリーを用いた定量化

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...