We thank Emily Mace and Malini Mukherjee for imaging and deconvolution analysis. Work in the Liu laboratory was supported in part by the Baylor-UTHouston Center for AIDS Research Core Support Grant number AI36211 from the National Institute of Allergy and Infectious Diseases, the Caroline Wiess Law Fund for Research in Molecular Medicine, Texas Children&#8217;s Hospital Pediatric Pilot Research Fund, and the Lymphoma SPORE Developmental Research Program from Baylor College of Medicine and the Methodist Research Institute.

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The authors have nothing to disclose.

現在の研究の新規性は、NK細胞のシナプスを研究するSTEDとSLBの技術を組み合わせることである。以前の研究は、T細胞のシナプス形成8を研究するためにTIRFと脂質二重層を撮像して、原形質膜6上の分子の輸送をシグナリングしている。他のものは、抗体でコーティングされたガラススライド13,14を使用してNK細胞のシナプスのSTED画像化を記載している。本明細書中にさらに記載のハイブリッド方法は、これらの脂質二重層の表面に超解像画像化によって得られる強化明確にNK細胞のシナプスを撮像する努力、より良いモデルAPCの動的表面上に構築。 のSLBは、実際の標的細胞またはAPCが有すること骨格、脂質ラフトを欠く人工膜、および他のリガンドであるが、この技術は、移動性およびリガンドの向きと重要な特徴を再現することができる。これはSLBシステムは解剖トンで還元主義的なアプローチとして機能することができますISとISのダイナミクスの形成に個々の受容体およびリガンドの寄与彼。のSLBの最も重要な特徴は、研究者はそのような共焦点及びTIRF顕微鏡法のような高分解能イメージング手法を使用して、この手法を組み合わせることができることである。 STED顕微鏡の導入は、さらに研究とその臨床応用がISに前例のない洞察を提​​供し、この利点を増大させる。 このシステムの一つの潜在的な批判は、SLBが十分に得られ、したがってシナプスにおける潜在的に非生理的解剖学的特徴を生じさせる、APCの複雑な表面を模倣しないことである。それがSLB上の表面分子の限られたレパートリーは完全にAPCの不均一人口​​表面を再現しないことは事実ですが、それがシナプス形成上の個々の受容体とリガンドの相互作用の影響を決定するために研究者を可能にするという点で、この制限は、有利であることができる。 Tここでの処理のいくつかの重要なステップがあります。最も重要なの中でのリポソームの酸化は、常にこのようなステップ1.10、2.6のように、チューブ、溶液中の酸素を置換するために、アルゴンを用いて、2.11を介して防止されることである。脂質の酸化は、このように自由に移動し、シナプスの構造に関与する表面タンパク質の能力を阻害、減少、脂質移動度をもたらす。同様に、凍結乾燥（ステップ1.2）により、リポソーム内のすべてのクロロホルムを除去することも重要です。脂質二重層中のタンパク質濃度の測定では、最初のクラスタのない均質な懸濁液にシリコンビーズを分散させるために重要である。必要であれば、ビーズの超音波処理を適用することができる。カバースリップを洗浄されるSLB、初期段階（5.1〜）を組み立てる際に、液滴が配置され、カバースリップは不可欠で固定されている。これらのいずれかでミスが（セクション5の先頭から）上で実験を開始する必要ができます。この理由のため、それは事故の場合には時間を節約するために必要とされるよりも多くのカバーガラスをきれいにすることをお勧め。 このシステムで作業しているとき、非クラスタは、最も頻繁な問題である。最後のステップで細胞を可視化する際に、1は任意の蛍光シナプスを見つけられなかった場合は、撮影することができいくつかのステップがあります。別のコグネイト細胞表面受容体のための染色細胞をガラスカバースリップまたは二重層の表面に非特異的に付着することができるが、細胞は重層とシナプスを形成していないことを確認するために、チャンバーに添加することができ、シナプス-関与する表面タンパク質べき非係合表面タンパク質は、細胞の周囲に拡散染色のように表示されます。一方で、細胞 - 二重層界面の面でのように明確なクラスタを表示されます。このメソッドは失敗した場合、1は、特定のマーカー1は、細胞表面上に十分な豊富に発現して勉強したいと考えていることを確実にするために、フローサイトメトリーを経由して 、それらの細胞を確認する必要があります。一定の表面proteインは、インビボ培養において長期間にわたり下方調節されることが知られている。 NK細胞のシナプス形成を視覚化する方法を具体的に、このプロトコルの詳細が、SLBシステムは単に、ステップ5.11において主リガンドの置換によって想像任意の免疫細胞におけるシナプス形成を研究するために使用することができる。複数のリガンドはまた、同時に添加してもよい。一つには、二重層内表面タンパク質を付着させるためのストレプトアビジン - ビオチン系を使用する必要はありません。ニッケルNTA：ヒスチジン相互作用も生存可能である。しかし、ストレプトアビジンの高強度と特異性に起因する：ビオチン相互作用、私たちの研究室ではこのシステムを好む。一つには、ステップ5.13において、所定濃度の二重層上に添加した細胞の濃度、ならびに成熟の異なる段階でシナプスを観察するために、その後のインキュベーション期間の持続時間を変えることができる。もちろん、これは細胞内のSTRを可視化する可能性を排除が、これはあっても、生きて行うことができますuctures（これらはすでに融合した蛍光タグで標識されていない限り、私たちの研究室は、いくつかのような変更された細胞株を使用しています）。これにより、プロトコルの可能な高度なカスタマイズのために、一つの基本的な脂質ダイナミクス15、シナプス形成を含む、免疫学、細胞生物学、および生化学における質問の非常に多様な範囲に対処するために、STEDイメージングと共に、基本SLB技法を使用することができ16、17細胞内シグナル伝達、および腫瘍細胞の転移18。

免疫学的シナプスは（IS）細胞の活性化と機能1の臨界接合部である。これは、抗原提示および細胞性免疫が行われることにより、主要な媒体である。シナプス形成の最も初期の顕微鏡研究は、細胞-細胞複合体システム2を利用した。このアプローチの主要な制限は、それがあったとして、複合体のほとんどは、このようにシナプスの構造自体の観察者の視界を制限する、「プロファイルで「見られることである。 1999年には、ダスティンの研究室では、以前のMcConnelラボ4,5によって開拓されていたガラスサポート脂質二重層（SLB）技術3を利用することによって、この制限に対処。このアプローチは、タンパク質が結合し、二次元で自由に移動することができたにガラスサポート脂質平面表面を支持する抗原提示細胞（APC）、処分した。この方法を使用して、ダスティンと同僚は、Tに直接バックアップピアすることができました彼は、高解像度の蛍光顕微鏡を用いて、初めてISの構造の「対面」外観を得るシナプス。 SLBシステムを用いて、ISを視覚化することが可能な詳細は、現在の画像化技術6-8の限界によって制限されている。標準照射技術を用いて、最小分解能（これらは区別することができる、請求つの異なるオブジェクト間、即ち、最小距離）は、レイリーの基準9に基づいて&lt;200nmであった。この制限は、シナプスを構成する非常に微細な、分子スケールの構造の画像化を妨害し、超解像技術の開発10-12まで、これらの構造の可視化は、電子顕微鏡を用いて固定された細胞の画像化に限定された。 そのようなSIMのような超解像技術は、種々の最近の出現により、（構造化照明顕微鏡）、PALM（光活性化ローカライゼーション顕微鏡）、STORM（stochastical光学復興顕微鏡）、およびSTED 10-12は 、研究者らは現在、順番にISのますます明確に理解を提供してきたこれまでにない詳細にこれらのシナプスの構造を研究することができます。 STED顕微鏡法の利点は、13の前に記載されている。ここでは、新たに開発された660nmの枯渇レーザーを備えたSTED顕微鏡で超解像画像を記述する。従来の592 nmの枯渇レーザーと比較して、660 nmのレーザーは、蛍光色素（http://nanobiophotonics.mpibpc.mpg.de/old/dyes/を参照）、特に、これらの赤色蛍光体のより広い選択が可能になる。 他の刊行物は、抗体でコーティングされたスライドガラス13,14にNK細胞のシナプスのSTED画像化を記載している。ここで、SLBシステムはNK細胞のシナプスを研究するために、超解像STED顕微鏡法と組み合わされる。この技術は、抗体を上回る利点を有している埋め込まれた表面タンパク質が平坦二次元平面（xy平面）内で自由に移動できる流動モザイクという被覆スライド。これは、より忠実に標的細胞の有機及び移動表面を模倣し、その結果、優れた生理学的に関連する免疫シナプスの形成を再現する。 このプロトコルの目的は、SLBシステムと、超解像STED顕微鏡法を組み合わせることにより、NK細胞のどの画像に免疫学的シナプスの詳細な説明と、エンドユーザに提供することである。それに必要な手順をエンドユーザに提供する：リポソームを調製し、タンパク質包埋二重層を構成する脂質二重層にタンパク質濃度を決定し、STED顕微鏡を用いて超解像画像を取得する。これらの技術は、免疫学の分野に限らず、広く、様々な分野にわたって使用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="1330">
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			<td height="21" style="height:21px;width:283px;">Name of Reagent/ Equipment</td>
			<td style="width:256px;">Company</td>
			<td style="width:316px;">Catalog Number</td>
			<td style="width:475px;">Purpose</td>
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			<td height="60" style="height:60px;width:283px;">18:1 (&#916;9-Cis) PC (DOPC) 
			1,2-dioleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:256px;">Avanti&#160;</td>
			<td style="width:316px;">850375C</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:283px;">18:1 Biotinyl Cap PE 
			1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt)</td>
			<td style="width:256px;">Avanti&#160;</td>
			<td style="width:316px;">870273C&#160;</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Argon gas, compressed</td>
			<td style="width:256px;">Airgas&#160;</td>
			<td style="width:316px;">UN1006</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">A lyophilizer</td>
			<td style="width:256px;">Labconco&#160;</td>
			<td style="width:316px;">Freezone 7740020</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Lyophilizer tubes</td>
			<td style="width:256px;">Labconco&#160;</td>
			<td style="width:316px;">7540200</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Chromatography Columns</td>
			<td style="width:256px;">Santa Cruz</td>
			<td style="width:316px;">sc-205558</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">1 M Tris pH 8.0</td>
			<td style="width:256px;">Ambion&#160;</td>
			<td style="width:316px;">AM9856</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5 M NaCl</td>
			<td style="width:256px;">Ambion&#160;</td>
			<td style="width:316px;">AM9759</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Octyl-&#946;-D-glucopyranoside</td>
			<td style="width:256px;">Sigma-Aldrich&#160;</td>
			<td style="width:316px;">O1008</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Dialysis tubing</td>
			<td style="width:256px;"><a href="http://www.spectrumlabs.com/generic/tms.html?LiFrom=%2Fdialysis%2FRCtubing.html;FrName=Spectra%2FPor+RC+Tubing;rtm=Kit;#KIT">Spectrum Labs&#160;</a></td>
			<td style="width:316px;">132676</td>
			<td>Liposome preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">96-well V-bottom polystyrene plate (untreated)</td>
			<td style="width:256px;">Corning</td>
			<td style="width:316px;">3896</td>
			<td>antibody density determination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Non-functionalized silica beads</td>
			<td style="width:256px;">Bangs Laboratories&#160;</td>
			<td style="width:316px;">SS06N</td>
			<td>antibody density determination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">FACS tubes</td>
			<td style="width:256px;">Fisher Scientific&#160;</td>
			<td style="width:316px;">14-959-2A&#160;</td>
			<td>antibody density determination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">QuantumTM MESF beads</td>
			<td style="width:256px;">Bangs Laboratories</td>
			<td style="width:316px;">Variable</td>
			<td>antibody density determination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Casein&#160;</td>
			<td style="width:256px;">Sigma-Aldrich&#160;</td>
			<td style="width:316px;">C0875</td>
			<td>Lipid bilayer preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">HEPES buffered saline + 1% HSA</td>
			<td style="width:256px;">Homemade</td>
			<td style="width:316px;"></td>
			<td>Lipid bilayer preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Streptavidin&#160;</td>
			<td style="width:256px;">Life technologies&#160;</td>
			<td style="width:316px;">434301</td>
			<td>Lipid bilayer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Fluorescently-labeled biotinylated protein&#160;</td>
			<td style="width:256px;">Homemade</td>
			<td style="width:316px;"></td>
			<td>Lipid bilayer preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">ibidi Sticky-Slide VI 0.4</td>
			<td style="width:256px;">ibidi&#160;</td>
			<td style="width:316px;">80608</td>
			<td>Lipid bilayer preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">25x75 mm glass coverslip</td>
			<td style="width:256px;">ibidi&#160;</td>
			<td style="width:316px;">10812</td>
			<td>Lipid bilayer preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Hydrogen peroxide (30%)</td>
			<td style="width:256px;">Fisher Scientic&#160;</td>
			<td style="width:316px;">BP2633</td>
			<td>Lipid bilayer preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Sulfuric acid</td>
			<td style="width:256px;">Sigma-Aldrich&#160;</td>
			<td style="width:316px;">258105</td>
			<td>Lipid bilayer preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">D-Biotin</td>
			<td style="width:256px;">Invitrogen&#160;</td>
			<td style="width:316px;">B20656</td>
			<td>Lipid bilayer preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Lens paper</td>
			<td style="width:256px;">VWR&#160;</td>
			<td style="width:316px;">54826-001</td>
			<td>For imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Type F Immersion Oil</td>
			<td style="width:256px;">Leica&#160;</td>
			<td style="width:316px;">11 513 859</td>
			<td>For imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">A Leica TCS STED microscope</td>
			<td style="width:256px;">Leica</td>
			<td style="width:316px;"></td>
			<td>For imaging</td>
		</tr>
	</tbody>
</table>

super-resolution imaging of the natural killer cell immunological synapse on a glass-supported planar lipid bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

図1は、脂質二重層上の抗体密度の結果を示す。原理は、フローサイトメトリー（A） を介して MFI対MESFの標準曲線を作成するための標準的なビーズを使用することである。サンプル列のMFIは、標準曲線を用いてMESFに変換した。脂質二重層上の抗体密度は、直線的に抗体濃度（B）と相関している。 図2ガラス支持平面脂質二重層におけるNKシナプスの三色STED画像を示す。脂質二重層上の抗CD16抗体は、Fアクチン形成及び分極およびNK細胞における免疫シナプスの焦点パネルでFアクチンメッシュパーフォリンの浸透を誘発する、蓄積する。この組み合わせたアプローチを使用して、1はきれいに直接NK細胞上のCD16のクラスタリングを反映しSLB、内抗CD16蛍光標識のマイクロクラスタを観察することができます。従来の共焦点画像を、CD16セントラの構造と比較してlのクラスタは、より容易により枯渇周囲の蛍光にSTED画像において識別される。また、アクチン細胞骨格の微細構造が大幅に向上解像度で見られる。以前の観察16,17と一致して、パーフォリン陽性溶菌顆粒はSTED画像、大部分は共焦点画像で失われている重要な詳細に低F-アクチン密度の領域の上に配置見られている。 <img alt="図1" src="/files/ftp_upload/52502/52502fig1highres.jpg" /> 脂質二重層における3G8抗体の図1の密度。（A）は 、標準シリーズのMESFおよびMFIとの間の線形相関。（B）のタンパク質濃度及び試料タンパク質の希釈系列の濃度と線形相関当たり蛍光標識されたタンパク質のモノマーの数を示す脂質でコーティングされた済州に増加する濃度の関数としての単位面積LICAビーズ。 <img alt="図2" src="/files/ftp_upload/52502/52502fig2highres.jpg" /> 平面脂質二重層におけるNKシナプスの図2 STEDイメージング。初代NK細胞は、SLBを含むビオチン化、蛍光標識された抗CD16（赤）で刺激した固定、透過処理し、その後ファロイジン（青色）および抗Fアクチンで染色した（緑色）。個々のセルは、第1、通常の共焦点設定で画像化され、その後、STED設定した。共焦点とSTED画像はホイヘンスソフトウェアを使用してデコンボリューションされた。スケールバーは1μm。 <a href="https://www.jove.com/files/ftp_upload/52502/52502fig2large.jpg" target="_blank">この図の拡大版については、ここをクリックしてください。</a>

Watch this Scientific Journal Video about Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer at JoVE.com

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

グラスサポート平面脂質二重層上のナチュラルキラー細胞免疫シナプスの超解像イメージング

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

super-resolution-imaging-natural-killer-cell-immunological-synapse-on

Research

JoVE Journal

Immunology and Infection

12.2K ビュー.  Texas Children's Hospital. We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

ビデオ: Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the flexibility of a VA approach in the analysis of biological datasets. Examples of these datasets in immunology include flow cytometry, Luminex data, and genotyping (e.g., single nucleotide polymorphism) data. Contrary to the traditional information visualization approach, VA restores the analysis power in the hands of analyst by allowing the analyst to engage in real-time data exploration process. We selected the VA software called Tableau after evaluating several VA tools. Two types of analysis tasks analysis within and between datasets were demonstrated in the video presentation using an approach called paired analysis. Paired analysis, as defined in VA, is an analysis approach in which a VA tool expert works side-by-side with a domain expert during the analysis. The domain expert is the one who understands the significance of the data, and asks the questions that the collected data might address. The tool expert then creates visualizations to help find patterns in the data that might answer these questions. The short lag-time between the hypothesis generation and the rapid visual display of the data is the main advantage of a VA approach.

Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.

ビジュアル分析手法と免疫学的データの解析を容易に

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

免疫・感染学

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

ナチュラルキラー細胞傷害性アッセイにおけるHIV - 1感染プライマリCD4 + T細胞を標的細胞としての準備と使用

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

合成脂質二重膜上でHIV-1エンベロープ誘発性ウイルス学的シナプスとシグナリングのイメージング

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

ナチュラルキラーT細胞の細胞（AAPC）媒介活性化と増殖を提示する人工抗原

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

デュアルカラータイム·ゲーティッド誘導放出枯渇（STED）ナノスによる免疫シナプスの可視化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

イメージング免疫学的シナプスダイナミクス内皮平面内細胞モデル

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

madSTORMを用いたT細胞の高度に多重化された超解像イメージング

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

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 ...

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, however, the lateral resolution of super-resolution imaging is only ~100 nm. The low resolution is mainly due to the high autofluorescence background of photosynthetic cells caused by high-intensity lasers that are required for super-resolution imaging, such as stochastic optical reconstruction microscopy (STORM). Here, we describe a photobleaching-assisted STORM method which was developed recently for imaging the marine picocyanobacterium Prochlorococcus. After photobleaching, the autofluorescence of Prochlorococcus is effectively reduced so that STORM can be performed with a lateral resolution of ~10 nm. Using this method, we acquire the in vivo three-dimensional (3-D) organization of the FtsZ protein and characterize four different FtsZ ring morphologies during the cell cycle of Prochlorococcus. The method we describe here might be adopted for the super-resolution imaging of other photosynthetic organisms.

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus

Here we describe a photobleaching method to reduce the autofluorescence of cyanobacteria. After photobleaching, stochastic optical reconstruction microscopy is used to obtain three-dimensional super-resolution images of the cyanobacterial FtsZ ring.

退色により、シアノ バクテリアプロクロロコッカスFtsZ リングの超解像イメージング

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, ...