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="http://www.jove.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...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH300070.03</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>1-Step Polymorphs</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>AN221725</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
<td>Remel/Fisher Scientific</td>
<td>R30852801</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human IL-2</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>202-IL</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human IL-15</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>247-IL</td>
<td>&nbsp;</td>
<tr>
<td>Protein G</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>19459</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human ICAM-1/Fc</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>720-IC</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human SDF-1</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>350-NS</td>
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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

Research

JoVE Journal

Immunology and Infection

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

46.1K 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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

Modification piggyBac système transposon des cellules T humaines primaires

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Recherche

Immunologie et infection

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 ...

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&amp;#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 ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Imagerie CD4 T Cellule interstitielle Migration dans les Derme Enflammé

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

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 ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolement et cytométrie de flux Analyse de Human Endocervical Gamma Delta cellules T

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolement et la Culture In Vitro de Macrophages alvéolaires murines et humaines

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Analyse de cisaillement induite par le flux de Migration des cellules B Zone marginale murin In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...