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.

La nouveauté de l&#39;étude est qu&#39;il combine la technique SLB avec STED pour étudier synapses des cellules NK. Des études antérieures ont imager la bicouche lipidique avec TIRF pour étudier des cellules T 8 la formation des synapses et la traite molécule de signalisation sur la membrane plasmique 6. D&#39;autres ont décrit STED imagerie des synapses des cellules NK en utilisant des lames de verre recouvertes d&#39;anticorps 13,14. Procédé hybride décrit ici en outre se appuie sur ces efforts par imagerie de la synapse des cellules NK avec la pureté améliorée conférée par imagerie super-résolution sur la surface de la bicouche lipidique, qui meilleurs modèles dynamique de la surface d&#39;une APC. Bien que des membranes artificielles SLBS sont dépourvues de cytosquelette, les radeaux lipidiques et d&#39;autres ligands que les cellules cibles réelles ou des APC possèdent, cette technique peut récapituler caractéristiques importantes telles que la mobilité et de l&#39;orientation de ligands. Cela permet au système SLB pour servir une approche réductionniste à disséquer til contribution des récepteurs et des ligands individuels à la formation de l&#39;IS et de la dynamique des IS. La caractéristique la plus importante de SLBS est que les chercheurs peuvent combiner cette technique avec des approches d&#39;imagerie à haute résolution, comme la microscopie confocale et la FRBR. L&#39;introduction de la microscopie STED augmente encore cet avantage, fournissant un aperçu sans précédent en est la recherche et de ses applications cliniques. Une critique potentiel de ce système est que le SLB ne reproduit pas de manière adéquate la surface d&#39;une APC complexe, donnant ainsi lieu à des caractéristiques anatomiques potentiellement non physiologiques dans les synapses qui en résultent. Se il est vrai que le répertoire limité de molécules de surface sur le SLB ne récapituler pas entièrement la surface hétérogène peuplé d&#39;une APC, cette limite peut également être avantageuse en ce qu&#39;elle permet aux chercheurs de déterminer l&#39;influence des interactions des récepteurs et ligands individuels sur la formation des synapses . Tvoici plusieurs étapes cruciales dans le processus. Parmi les plus critiques, ce est que l&#39;oxydation des liposomes être empêchée par l&#39;utilisation de l&#39;argon constamment à déplacer l&#39;oxygène dans le tube et une solution, par exemple dans les étapes 2,6, 1,10, et 2,11. L&#39;oxydation des lipides se traduira par une diminution de la mobilité des lipides, ce qui empêche ainsi la capacité des protéines de surface de se déplacer librement et de participer à la structuration synaptique. De même, il est également crucial d&#39;enlever tout le chloroforme dans le liposome par lyophilisation (étape 1.2). Dans la détermination de la densité de la protéine dans la bicouche lipidique, il est d&#39;une importance de premier disperser les perles de silicium en une suspension homogène exempt de groupes. Si nécessaire, la sonication de billes peut être appliquée. Dans le montage de la SLB, les premières étapes (05.01 à 05.08), dans lequel les lamelles sont nettoyés, les gouttes sont placées, et lamelle est apposée sont vitales. Une erreur dans l&#39;un de ces peut nécessiter de commencer l&#39;expérience plus (depuis le début de l&#39;article 5). Pour cette raison, il estbonnes pratiques pour nettoyer plus de lamelles sera nécessaire pour gagner du temps en cas d&#39;accident. Non-regroupement est la question la plus fréquente lorsque l&#39;on travaille avec ce système. Si, lors de la visualisation des cellules dans l&#39;étape finale, on ne parvient pas à trouver des synapses fluorescentes, il ya quelques étapes qui peuvent être prises. Une autre tache pour le récepteur de surface cellulaire apparenté peut être ajouté à la chambre afin de vérifier que la cellule n&#39;a pas formé une synapse avec la bicouche, Alors que les cellules peuvent adhérer de manière non spécifique à la surface de la lamelle de verre ou bicouche, les protéines de surface synaptique-impliquées devraient apparaît comme grappes distinctes au niveau du plan de l&#39;interface cellule-bicouche, tandis que les protéines de surface non engagés devraient apparaître comme coloration diffuse autour du périmètre de la cellule. Si cette méthode échoue, il faut vérifier leurs cellules par cytométrie de flux pour assurer que le marqueur particulier une espère étudier est exprimée en abondance suffisante sur la surface de la cellule. Certains prote de surfaceins sont connus pour être régulé à la baisse plus long terme dans la culture vivo. Bien que ce protocole détails spécifiquement comment NK visualiser la formation des synapses des cellules, le système SLB peut être utilisée pour étudier la formation des synapses dans toute immunocytes imaginable simplement par la substitution du ligand dans l&#39;étape primaire 5,11. Plusieurs ligands peuvent également être ajoutés simultanément. Un devez également pas utiliser un système streptavidine-biotine pour faire adhérer les protéines de surface dans la bicouche. Nickel-NTA: interactions histidine sont également viable. Toutefois, en raison de la résistance élevée et la spécificité de la streptavidine: interaction biotine, notre laboratoire préfère ce système. On peut également faire varier la concentration de cellules ajoutées sur la bicouche de la densité prédéterminée à l&#39;étape 5.13, ainsi que la durée de la période d&#39;incubation subséquente afin d&#39;observer les synapses à différents stades de maturation. Cela peut même être fait en direct, bien que cela exclut évidemment la possibilité de visualiser str intracellulaireuctures (sauf se ils sont déjà étiquetés avec une étiquette fluorescente fusionnée; notre laboratoire utilise quelques telles lignées cellulaires modifiées). En raison du degré élevé de personnalisation possible dans ce protocole, on peut utiliser la technique SLB de base, ainsi que l&#39;imagerie STED, pour répondre à un éventail incroyable diversité de questions en immunologie, la biologie cellulaire et la biochimie, y compris la dynamique de lipides de base 15, la formation des synapses 16, 17 signalisation intracellulaire, et la cellule tumorale 18 métastases.

La synapse immunologique (IS) se trouve à un carrefour critique pour l&#39;activation et la fonction une cellule. Ce est le moyen principal par lequel la présentation des antigènes et de l&#39;immunité à médiation cellulaire sont réalisés. Les premières études microscopiques de la formation des synapses utilisés un système conjugué cellule-cellule 2. La principale limite de cette approche est que la plupart des conjugués sera consulté &#39;profil&#39;, pour ainsi dire, limitant ainsi le point de vue de l&#39;observateur de la structure synaptique lui-même. En 1999, le laboratoire Dustin adressée cette limitation en utilisant la technique 3-verre soutenue bicouche lipidique (SLB), qui avait été un pionnier tôt par le laboratoire 4,5 McConnel. Cette approche disposé de cellules présentatrices d&#39;antigène (CPA) en faveur d&#39;une surface plane lipidique verre soutenue, dans lequel protéines pourraient être attachés et se déplacent librement dans deux dimensions. En utilisant cette méthode, Dustin et ses collègues ont pu pairs directement vers le haut dans til Synapse utilisant la microscopie à fluorescence haute résolution, et pour la première fois obtenir un look &quot;face-à-face&quot; à la structure de l&#39;IS. Avec l&#39;utilisation du système SLB, le détail avec lequel la SI peut être visualisé a été limité seulement par les limites des techniques actuelles d&#39;imagerie 8.6. En utilisant des techniques d&#39;éclairage classiques, la résolution minimale (ce est à dire, la distance minimale entre deux objets distincts dans laquelle ils peuvent être distingués) a été &lt;200 nm sur la base du critère de Rayleigh neuf. Cette limite empêche l&#39;imagerie de structures très fines, à l&#39;échelle moléculaire qui composent la synapse, et jusqu&#39;à ce que le développement de techniques d&#39;imagerie de super-résolution 12/10, la visualisation de ces structures se est limitée à l&#39;imagerie de cellules fixées à l&#39;aide de la microscopie électronique. Avec l&#39;avènement récent d&#39;une variété de techniques de super-résolution, telles que la carte SIM (microscopie éclairage structuré), PALM (microscopie de localisation photoactivé), STORM (microscopie de reconstruction optique stochastique) et STED 10-12, les enquêteurs sont maintenant en mesure d&#39;étudier ces structures synaptiques en détail sans précédent, qui a à son tour fourni une compréhension de plus clarifié de l&#39;IS. Les avantages de la microscopie STED ont été décrits avant le 13. Nous décrivons ici super-résolution imagerie par microscopie STED équipé du laser 660 nm d&#39;épuisement nouvellement développé. Par rapport à la 592 nm laser appauvrissement classique, le laser de 660 nm permet une sélection plus large de colorants fluorescents (voir http://nanobiophotonics.mpibpc.mpg.de/old/dyes/), en particulier ces fluorophores rouges. Autres publications ont décrit l&#39;imagerie STED des synapses des cellules NK sur des lames de verre recouvertes d&#39;anticorps 13,14. Ici, le système est combiné avec SLB super-résolution microscopie STED pour étudier la cellule NK synapse. Cette technique présente l&#39;avantage par rapport aux anticorps-des lames revêtues d&#39;être une mosaïque de fluide, dans lequel les protéines de surface embarqués peuvent se déplacer librement dans une surface à deux dimensions à plat (plan xy). Cela imite plus fidèlement la surface organique et mobiles d&#39;une cellule cible, et par conséquent mieux récapitule la formation d&#39;une synapse immunologique physiologiquement pertinent. L&#39;objectif de ce protocole est de fournir à l&#39;utilisateur final avec une description détaillée de la façon de l&#39;image de la synapse immunologique des cellules NK en combinant le système SLB et de super-résolution STED microscopie. Il fournira à l&#39;utilisateur final avec les mesures nécessaires pour: préparer les liposomes, construire bicouches protéines intégré, déterminer la densité de protéine sur les bicouches lipidiques, et acquérir des images de super-résolution utilisant la microscopie STED. Ces techniques ne sont pas limités au domaine de l&#39;immunologie, et peuvent être largement utilisés dans divers domaines.

<table border="0" cellpadding="0" cellspacing="0" width="1330">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="60">
			<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.

La figure 1 montre le résultat de la densité de l&#39;anticorps sur la bicouche lipidique. Le principe est d&#39;utiliser des billes standard pour rendre la courbe standard du MESF par rapport IMF par cytométrie de flux (A). Le MFI de la série d&#39;échantillons a été converti en MESF en utilisant la courbe standard. La densité d&#39;anticorps sur la bicouche lipidique est linéairement corrélée avec la concentration d&#39;anticorps (B). Figure 2 montrent la couleur triple STED l&#39;image de NK synapse sur verre-support plan bicouche lipidique. L&#39;anticorps anti-CD16 sur la bicouche lipidique se accumule, provoquant la formation de F-actine et de la polarisation et de la pénétration à travers les mailles perforine F-actine à partir du panneau central de synapse immunologique des cellules NK. En utilisant cette approche combinée, on peut observer les microamas proprement marquées par fluorescence de anti-CD16 dans le SLB, qui reflète directement le regroupement de CD16 sur la cellule NK. Par rapport à l&#39;image confocale classique, la structure du CD16 central cluster est plus facilement discerné dans l&#39;image la STED en raison de la fluorescence ambiante appauvri. En outre, l&#39;ultrastructure du cytosquelette d&#39;actine est vu avec une résolution améliorée de manière significative. Conformément aux observations antérieures 16,17, les granules lytiques perforine positif sont considérés positionné au-dessus des régions de faible densité F-actine dans l&#39;image la STED, un détail crucial qui est la plupart du temps perdu dans l&#39;image confocale. <img alt="Figure 1" src="/files/ftp_upload/52502/52502fig1highres.jpg" /> Figure 1. Densité d&#39;anticorps 3G8 sur bicouche lipidique. (A) de corrélation linéaire entre MESF et IMF pour la série standard. (B) corrélation linéaire entre la densité des protéines et de la concentration pour la série protéine échantillon de dilution, indiquant le nombre de monomères marquées par fluorescence protéines par unité de surface en fonction de la concentration croissante de Si revêtus de lipidesperles de lica. <img alt="Figure 2" src="/files/ftp_upload/52502/52502fig2highres.jpg" /> Figure 2. STED imagerie de NK synapse sur plan bicouche lipidique. Cellules NK primaire ont été stimulées sur le SLB contenant biotinylé marqué par fluorescence anti-CD16 (rouge), fixées, perméabilisées, puis colorées avec la phalloïdine (bleu) et anti-F-actine (vert). Une cellule individuelle a été imagé sous le paramètre confocal normale, puis le réglage STED. Images confocale et STED ont été déconvolution en utilisant un logiciel Huygens. Barre d&#39;échelle, une um. <a href="https://www.jove.com/files/ftp_upload/52502/52502fig2large.jpg" target="_blank">Cliquez ici pour une version plus grande de cette figure.</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.

Super-résolution de l&#39;imagerie cellulaire Synapse immunologique Natural Killer sur un verre soutenu par Planar lipidique en deux couches

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 vues.  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.

Vidéo: 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.

Faciliter l&#39;analyse des données immunologiques avec Visual techniques analytiques

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

Recherche

Immunologie et infection

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.

Préparation et utilisation de VIH-1 primaires CD4 + T-cellules comme cellules cibles dans des cellules tueuses naturelles Tests cytotoxiques

 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.

Imagerie de VIH-1 Enveloppe induite Synapse virologique et de signalisation sur bicouches lipidiques synthétiques

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.

Présentatrice d&#39;antigène artificielle d&#39;activation à médiation cellulaire (AAPC) et l&#39;expansion des cellules T tueuses naturelles

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.

Visualisation du immunologique Synapse par double couleur Temps-dépendants émission stimulée épuisement (STED) nanoscopie

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'.

Une cellule endothéliale Planar Modèle for Imaging Dynamics Synapse immunologique

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.

Cytométrie en flux Analyse des cellules tueuses naturelles lytique Activité dans le sang humain

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.

Imagerie haute résolution et ultra-résolution des cellules T à l&#39;aide de madSTORM

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.

Un nouveau système exempt d’alimentation pour la Production de masse des cellules tueuses naturelles murins 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 ...

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.

Photoblanchiment permet l’imagerie de l’anneau FtsZ dans la cyanobactérie Prochlorococcus Super-résolution

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