Ce projet a été soutenu par des subventions des National Institutes Health HL087088 (MK) et HL18208 (MK).

1. Isolement des lymphocytes T humains <ol><li> Obtenir le sang humain à partir d&#39;un donneur sain. Laisser le sang se refroidir à température ambiante (~ 30 minutes) avant de passer à l&#39;étape suivante. </li><li> Délicatement la pipette 3 ml de Métamorphose Salle des médias de température à gradient de densité dans un tube 8 ml en polystyrène à fond rond. Ajouter délicatement 3 mL de sang total sur le dessus du support Métamorphose. Il est important d&#39;éviter le mélange des deux réactifs. </li><li> Centrifuger les tubes à 500 g pendant 45 minutes à température ambiante. </li><li> Après la centrifugation, les cellules mononucléées du sang périphérique (PBMC) ont maintenant séparé de d&#39;autres composants sanguins dans la couche cellulaire supérieure. La couche de PBMC apparaît, de haut en bas, comme le premier groupe nuageux. </li><li> Retirer délicatement le clair de couleur jaune phase supérieure du sang, au-dessus de la couche PBMC, puis utiliser une micropipette P1000 à transférer la couche PBMC à un ml 15 ou 50 ml tube conique. </li><li> Laver les PBMC à deux reprises avec du PBS, les cellules de centrifugation à 500 xg pendant 5 minutes chaque fois. Le surnageant sera un peu nuageux après chaque lavage. </li></ol> 2. Culture de lymphocytes T humains <ol><li> Avec une pipette, transférer les PBMC d&#39;un flacon T-75 de la culture dans 20 ml du milieu RPMI 1640 contenant 10% de FBS, 1% de pénicilline / streptomycine et 1 pg / mL phytohémagglutinine (PHA). </li><li> Incuber à 37 ° C et 5% de CO 2 d&#39;au moins 1 heure, et jusqu&#39;à 24 heures. Cette étape permet de monocytes, qui sera adhérente à la surface de ballon, d&#39;être séparé de l&#39;lymphocytes qui restent en suspension. Si une incubation courte (1 heure) est utilisé à cette étape, il est acceptable d&#39;utiliser du milieu RPMI 1640 contenant 10% de FBS et 1% de pénicilline / streptomycine, sans complétant avec PHA comme spécifié à l&#39;étape 2.1. </li><li> Retirez soigneusement l&#39;ensemble des médias de la fiole, ajoutez-le à un tube conique de 50 ml, et centrifuger à 500 xg pendant 5 minutes. </li><li> Reprendre le culot cellulaire, qui contient principalement des lymphocytes aujourd&#39;hui, et le transfert des cellules d&#39;un nouveau T-75 flacon contenant 25 ml du milieu RPMI 1640 contenant 10% de FBS, 1% de pénicilline / streptomycine et 1 ug / ml de PHA. </li><li> Incuber à 37 ° C pendant 3 jours (2 jours si l&#39;incubation initiale des PBMC a été toute la nuit). Après 24 heures de croissance, il peut être nécessaire d&#39;ajouter 15-20 ml de milieux frais et le transfert à un plus grand T-175 flacon. </li><li> Après 3 jours, utiliser une pipette pour enlever les médias et les lymphocytes en suspension dans le ballon et le transfert d&#39;un tube conique de 50 ml. Centrifuger à 500 xg pendant 5 minutes. </li><li> Reprendre le culot cellulaire et des cellules de transfert à un nouveau flacon T-75 ou T-175 contenant 25 ml (T-75) ou 50 ml (T-175) RPMI 1640 avec 10% de FBS, 1% de pénicilline / streptomycine et 20 ng / ml IL-2 humaine ou l&#39;IL-15. </li><li> Cultivez les lymphocytes pendant 4-7 jours. Si en commençant par un flacon T-75, la culture devra être élargi et transféré dans un flacon T-175 après 1-2 jours. </li></ol> 3. Dosage in vitro la migration lymphocytaire <ol><li> 1 jour avant le test de migration, un manteau à fond de verre plat de 0,17 mm avec 20 pg / ml de protéine A ou G en PBS pendant une nuit à 4 ° C. </li><li> Laver abondamment le plat avec du PBS. </li><li> Immobiliser humaine ICAM-1/Fc (10 pg / ml) et humaines du SDF-1 (2 pg / ml) en ajoutant ces réactifs en solution PBS pour le plat et l&#39;incubation de 4 heures à température ambiante. </li><li> Préparer les lymphocytes T en déterminant d&#39;abord la densité des cellules en culture en flacon T-175 en utilisant un hématimètre. Environ 2-5 x 10 5 cellules doit être utilisé par plat pour atteindre une densité cellulaire appropriée pour l&#39;analyse de la migration. </li><li> Lavez les lymphocytes T à deux reprises avec du PBS, puis remettre en suspension dans 1 ml L-15 supports contenant 1 mg / ml de D-glucose. </li><li> Laver la vaisselle et les enduits de ICAM-1/Fc SDF-1 abondamment avec du PBS. </li><li> Transfert lymphocytes T dans les médias 1 mL de la vaisselle et les maintenir à 37 ° C. </li></ol> 4. Capture d&#39;une séquence d&#39;images en utilisant NIS éléments logiciels <ol><li> Ouvrez NIS Elements. </li><li> Dans la caméra du menu Paramètres, choisissez «binning 2x2&quot; que le mode à la fois pour l&#39;imagerie en direct et la capture d&#39;image. </li><li> Allez dans le menu Applications et choisissez Définir / Run Experiment. </li><li> Choisissez la durée de temps entre les images et la longueur totale de la séquence de capture d&#39;image. </li><li> Appuyez sur le bouton &quot;Exécuter&quot; pour commencer l&#39;acquisition des images. </li><li> Les cellules peuvent être suivis et la migration des paramètres (vitesse, longueur du trajet, les déplacements, etc) ont quantifié en utilisant une des plusieurs logiciels, dont ImageJ, AutoQuant ou Volocity (figure 1). </li><li> Pour générer un «complot toile d&#39;araignée&quot;, les coordonnées XY pour chaque cellule et timepoint sont collectées (figure 2) et projetée sur un graphique avec un point de départ commun pour chacune des cellules à l&#39;origine (figure 3). </li></ol> 5. Les résultats représentatifs Au jour 6 de la culture dans la présence de lymphocytes T soit IL-2 ou l&#39;IL-15,MAQUILLAGE&gt; 98% des cellules, tel que déterminé par coloration CD3 positifs ainsi que des CD4 positifs et / ou CD8 coloration. Pour l&#39;IL-2, nous avons trouvé 83% des CD4 +, les cellules CD8 + 15% et &lt;1% des CD4 + CD8 +. Pour l&#39;IL-15, nous avons trouvé 88% des CD4 +, les cellules CD8 + 11% et &lt;1% des CD4 + CD8 +. Pendant la migration lymphocytaire T sur ICAM-1/SDF-1 substrats, les cellules présentaient une vitesse d&#39;environ 15 um / min qui peut être maintenu sur une période de 1 heure. T sur la migration lymphocytaire ICAM-1/SDF-1 dépend de LFA-1 médiée par l&#39;adhésion, comme un anti-LFA-1 ligand bloquant anticorps inhibe fortement la migration. <img src="/files/ftp_upload/2017/2017fig1.jpg" alt="Figure 1" /> Figure 1. Cellule de suivi de la migration des lymphocytes T. Lymphocytes T isolés du sang total et cultivées en présence d&#39;IL-2 ou l&#39;IL-15 pendant 6 jours ont été autorisés à adhérer et migrer sur fond de verre plats revêtus d&#39;10 pg / mL ICAM-1 et 2 pg / mL SDF-1. Les images ont été acquises toutes les 10 secondes pendant 30 minutes. Les cellules ont été identifiés dans chaque image et suivis dans le temps en utilisant le logiciel Volocity. Ce film montre la migration aléatoire des lymphocytes T à une vitesse de ~ 15 um / min. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/2017/2017_Kim_Movie1.mov">Cliquez ici pour voir la vidéo.</a> Cellule de suivi Movie 1. Migration des lymphocytes T. Lymphocytes T isolés du sang total et cultivées en présence d&#39;IL-2 ou l&#39;IL-15 pendant 6 jours ont été autorisés à adhérer et migrer sur fond de verre plats revêtus d&#39;10 ug / ml d&#39;ICAM-1 et 2 pg / mL SDF-1 . Les images ont été acquises toutes les 10 secondes pendant 30 minutes. Les cellules ont été identifiés dans chaque image et suivis dans le temps en utilisant le logiciel Volocity. Ce film montre la migration aléatoire des lymphocytes T à une vitesse de ~ 15 um / min. <img src="/files/ftp_upload/2017/2017fig2.jpg" alt="Figure 2" /> Figure 2. Position de la cellule spatio-temporelle. Les coordonnées XY de chaque cellule ont été obtenues pour chaque point de temps en utilisant le logiciel Volocity. <img src="/files/ftp_upload/2017/2017fig3.jpg" alt="Figure 3" /> Figure 3. &quot;Spider Web intrigue.&quot; Utilisation de la XYT coordonnées de chaque cellule, 15 cellules ont été choisis au hasard et tracées avec un point de départ commun à l&#39;origine. En comparant des parcelles toile d&#39;araignée donne une représentation visuelle rapide des différences de migration entre les différentes conditions expérimentales. Parcelles à la migration lymphocytaire T dans des conditions de contrôle (à gauche) et en présence d&#39;un anti-LFA-1 ligand-anticorps bloquant (à droite).

<ol>
	<li>Hyun, Y. M., Chung, H. L., McGrath, J. L., Waugh, R. E., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+integrin+VLA-4+localizes+to+the+lamellipodia+and+mediates+t+cell+migration+on+VCAM-1.">Activated integrin VLA-4 localizes to the lamellipodia and mediates t cell migration on VCAM-1.</a> J Immunol. 183, 359-369 (2009).</li><li>Morin, N. A., Oakes, P. W., Hyun, Y. M., Lee, D., Chin, Y. E., King, M. R., Springer, T. A., Shimaoka, M., Tang, J. X., Reichner, J. S., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonmuscle+myosin+heavy+chain+IIA+mediates+integrin+LFA-1+de-adhesion+during+T+lymphocyte+migration.">Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.</a> The Journal of experimental medicine. 205, 195-205 (2008).</li><li>Hyun, Y. M., Lefort, C. T., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+integrins+and+their+ligand+interactions.">Leukocyte integrins and their ligand interactions.</a> Immunologic research. , (2009).</li></ol>

Dans cette expérience, nous fournissons des détails sur un système simple pour analyser la motilité des lymphocytes T primaires humains. Dans les essais de migration in vitro ont été utilisés pour disséquer les rôles de nombreuses molécules et voies de signalisation impliquées dans la locomotion des différents types cellulaires. Certains contrôles essentiels à garder à l&#39;esprit lorsque vous concevez votre propre expérience de notre protocole comprennent: 1) Non-adhésif ou non inté...

<table><tbody><tr><td>RPMI 1640</td><td>GIBCO, by Life Technologies</td><td>11875</td><td></td></tr><tr><td>FBS</td><td>Thermo Fisher Scientific, Inc.</td><td>SH300070.03</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>1-Step Polymorphs</td><td>Accurate Chemical &amp;amp; Scientific Corporation</td><td>AN221725</td><td></td></tr><tr><td>PHA</td><td>Remel, Thermo Fisher Scientific</td><td>R30852801</td><td></td></tr><tr><td>Rec. human IL-2</td><td>R&amp;amp;D Systems</td><td>202-IL</td><td></td></tr><tr><td>Rec. human IL-15</td><td>R&amp;amp;D Systems</td><td>247-IL</td><td></td></tr><tr><td>Protein G</td><td>Sigma-Aldrich</td><td>19459</td><td></td></tr><tr><td>Rec. human ICAM-1/Fc</td><td>R&amp;amp;D Systems</td><td>720-IC</td><td></td></tr><tr><td>Rec. human SDF-1</td><td>R&amp;amp;D Systems</td><td>350-NS</td><td></td></tr></tbody></table>

human t lymphocyte isolation, culture and analysis of migration in vitro

La migration des lymphocytes T implique l&#39;interaction adhésive des intégrines de surface des cellules avec des ligands exprimés sur d&#39;autres cellules ou avec des protéines de la matrice extracellulaire. L&#39;activation des intégrines spatiotemporelle précise d&#39;un état de faible affinité à un état de haute affinité au bord leader de cellules est important pour la migration des lymphocytes T<sup&gt; 1</sup&gt;. De même, la rétraction de la cellule de bord de fuite, ou uropode, est une étape nécessaire au maintien de la persistance de l&#39;intégrine-dépendante la motilité des lymphocytes T<sup&gt; 2</sup&gt;. De nombreuses approches thérapeutiques pour auto-immunes ou inflammatoires intégrines maladies cibles comme un moyen d&#39;inhiber le recrutement excessif et la migration des leucocytes<sup&gt; 3</sup&gt;. Afin d&#39;étudier les événements moléculaires qui régulent la migration des lymphocytes T humains, nous avons utilisé une<em&gt; In vitro</em&gt; Système d&#39;analyser la migration des cellules sur un substrat en deux dimensions qui imite l&#39;environnement que rencontre un lymphocyte T au cours de recrutement de la vascularisation. Les lymphocytes T sont d&#39;abord isolé à partir de donneurs humains et sont alors stimulées et mises en culture pendant sept à dix jours. Pendant le test, les lymphocytes T sont admises à adhérer et migrer sur un substrat revêtu avec la molécule-1 d&#39;adhésion intercellulaire (ICAM-1), un ligand pour intégrine LFA-1, et des cellules stromales-derived factor-1 (SDF-1). Nos données montrent que les lymphocytes T présentent une vitesse de migration des ~ 15 um / min. La migration des lymphocytes T peut être inhibée par le blocage de l&#39;intégrine<sup&gt; 1</sup&gt; Ou par des inhibiteurs de la machinerie cellulaire qui régule actomyosine migration cellulaire<sup&gt; 2</sup&gt;.

Watch this Scientific Journal Video about Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro at JoVE.com

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

La migration des lymphocytes T se produit pendant homing vers les organes lymphoïdes, la sortie de la vascularisation, et entrer dans les tissus périphériques. Ici, nous décrivons un protocole qui peut être utilisé pour analyser la migration lymphocytaire T<em&gt; In vitro</em&gt;.

Human T isolement des lymphocytes, de la Culture et de l&#39;analyse des migrations In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

human-t-lymphocyte-isolation-culture-analysis-migration

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

46.3K vues.  University of Rochester. La migration des lymphocytes T se produit pendant homing vers les organes lymphoïdes, la sortie de la vascularisation, et entrer dans les tissus périphériques. Ici, nous décrivons un protocole qui peut être utilisé pour analyser la migration lymphocytaire T In vitro.

Vidéo: Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Lymphocytes Isolation de la peau humaine pour Analyse phénotypique et<em&gt; Ex Vivo</em&gt; Culture cellulaire

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Recherche

Immunologie et infection

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Souris Naïf CD4<sup&gt; +</sup&gt; Isolement des cellules T et<em&gt; In vitro</em&gt; Différenciation en sous-ensembles de cellules T

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolement et activation de murins Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Analyse des Lymphocytes extravasation L&#39;utilisation d&#39;un<em&gt; In vitro</em&gt; Modèle de la barrière hématoencéphalique humaine

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Quantifier la chimiotaxie Monocyte humain In Vitro et lymphocytes murins traite In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

Microscopie de lumière-feuille pour la visualisation en trois dimensions des cellules immunitaires humaines

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Évaluation de la migration des lymphocytes dans un système de transmigration ex vivo

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Avancée d’imagerie du poumon Homing des Lymphocytes humains dans un modèle In Vivo de l’Inflammation allergique, issue des feuilles lumière microscopie expérimentale

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Médecine

Elucider la cinétique des lymphocytes T : méthodes d’analyse de migration en temps réel

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

Pleins feux sur l’auteur : Comprendre les mécanismes de la maladie grâce à l’analyse en temps réel de la migration des lymphocytes T

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

Essai de migration in vitro en temps réel pour les lymphocytes T CD8+ murins primaires

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

Human T isolement des lymphocytes, de la Culture et de l'analyse des migrations In Vitro

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Essai de migration in vitro en temps réel pour les lymphocytes T CD8+ murins primaires

Essai de migration in vitro en temps réel pour les lymphocytes T CD8+ murins primaires