Dieses Projekt wurde von den National Institutes of Health Grants HL087088 (MK) und HL18208 (MK) unterstützt.

1. Isolierung von humanen T-Lymphozyten <ol><li> Erhalten menschlichem Blut von einem gesunden Spender. Lassen Sie das Blut auf Raumtemperatur (~ 30 min), bevor Sie den nächsten Schritt zu kühlen. </li><li> Gently Pipette 3 ml Raumtemperatur Polymorph Dichtegradienten Medien in einer 8 ml-Polystyrol Röhre. Vorsichtig 3 ml Vollblut auf der Oberseite des Polymorph Medien. Es ist wichtig zur Vermeidung einer Vermischung der beiden Reagenzien. </li><li> Zentrifugieren bei 500 xg für 45 Minuten bei Raumtemperatur. </li><li> Nach der Zentrifugation wurden die peripheren mononukleären Blutzellen (PBMC) nun von anderen Blutbestandteilen in die oberste Zelle abgetrennt. Die PBMC-Schicht erscheint, von oben nach unten, als der erste Regen-Band. </li><li> Entfernen Sie vorsichtig die klare gelb-gefärbten oberen Phase des Blutes, über den PBMC-Schicht, und dann mit einem P1000 Mikropipette auf die PBMC-Schicht auf einer 15 ml oder 50 ml konische Röhrchen füllen. </li><li> Waschen Sie die PBMC zweimal mit PBS, Zentrifugation Zellen bei 500 xg für 5 Minuten jeder Zeit. Der Überstand wird etwas trübe nach jeder Wäsche. </li></ol> 2. Kultur von humanen T-Lymphozyten <ol><li> Mit einer Pipette die PBMC zu einer T-75 Kulturflasche in 20 ml RPMI 1640 Medium mit 10% FBS, 1% Penicillin / Streptomycin und 1 pg / mL Phytohämagglutinin (PHA). </li><li> Bei 37 ° C und 5% CO 2 für mindestens 1 Stunde und bis zu 24 Stunden. Dieser Schritt ermöglicht Monozyten, die sein Anhänger in den Kolben Oberfläche, von der Lymphozyten, die in der Schwebe bleiben getrennt werden. Wenn eine kurze Inkubationszeit (1 Stunde) bei diesem Schritt verwendet wird, ist es akzeptabel, RPMI 1640 Medium mit 10% FBS und 1% Penicillin / Streptomycin ohne Ergänzung mit PHA wie in Schritt 2.1 angegebene Verwendung. </li><li> Entfernen Sie vorsichtig alle Medien aus dem Kolben, fügen Sie ihn in einen 50 mL konischen Rohr, und Zentrifuge bei 500 xg für 5 Minuten. </li><li> Zellpellet, das nun in erster Linie enthält Lymphozyten, und übertragen Sie die Zellen, um eine neue T-75 Kolben mit 25 ml RPMI 1640 Medium mit 10% FBS, 1% Penicillin / Streptomycin und 1 pg / mL PHA. </li><li> Bei 37 ° C für 3 Tage (2 Tage, wenn die anfängliche Inkubation von PBMC wurde über Nacht). Nach 24 Stunden Wachstum, kann es notwendig sein, um 15-20 ml frisches Medium hinzufügen und Übertragung auf einen größeren T-175 Kolben. </li><li> Nach 3 Tagen Einsatz einer Pipette auf die Medien und suspendiert Lymphozyten aus dem Kolben und in ein 50 mL konischen Rohr zu entfernen. Zentrifuge bei 500 xg für 5 Minuten. </li><li> Zellpellet und Transfer-Zellen zu einem neuen T-75 oder T-175 Kolben mit 25 ml (T-75) oder 50 ml (T-175) RPMI 1640 mit 10% FBS, 1% Penicillin / Streptomycin und 20 ng / mL humanem IL-2 oder IL-15. </li><li> Wachsen Lymphozyten für 4-7 Tage. Wenn beginnend mit einem T-75-Kolben, wird die Kultur müssen ausgebaut und in ein T-175 Kolben nach 1-2 Tagen. </li></ol> 3. In Vitro Lymphozytenmigration Assay <ol><li> 1 Tag vor der Migration Assay Fell einen Glasboden 0,17 mm Schale mit 20 ug / mL Protein A oder G in PBS über Nacht bei 4 ° C. </li><li> Waschen Sie die Teller ausgiebig mit PBS. </li><li> Immobilisieren menschlichen ICAM-1/Fc (10 ug / mL) und der menschlichen SDF-1 (2 pg / mL), indem Sie diese Reagenzien in PBS-Lösung in die Schale und Inkubation 4 Stunden bei Raumtemperatur. </li><li> Bereiten Sie T-Lymphozyten, indem zunächst die Zelldichte in der Kultur in der T-175 Kolben mit einem Hämazytometer. Etwa 2-5 x 10 5 Zellen pro Schale sollte genutzt werden, um eine Zelldichte geeignet für Migration Analyse zu erreichen. </li><li> Wash T-Lymphozyten zweimal mit PBS und anschließend in 1 ml L-15 Medien mit 1 mg / mL D-Glucose resuspendieren. </li><li> Waschen Sie die Teller mit ICAM-1/Fc und SDF-1 intensiv mit PBS beschichtet. </li><li> Transfer-T-Lymphozyten in 1 ml Medium in die Schale und halten sie bei 37 ° C. </li></ol> 4. Aufnehmen eines Bilds Sequence mit NIS Elements Software <ol><li> Öffnen Sie NIS Elements Software. </li><li> In die Kamera-Einstellungen Wählen Sie im Menü &quot;2x2 Binning&quot;, wie der Modus für Live-Imaging-und Bilderfassung. </li><li> Zum Menü Anwendungen und wählen Sie Definieren / Run Experiment. </li><li> Wählen Sie die Länge der Zeit zwischen den Bildern und die Gesamtlänge der Bilderfassung Sequenz. </li><li> Drücken Sie die Schaltfläche &quot;Ausführen&quot; die Bildaufnahme beginnen. </li><li> Die Zellen können verfolgt und Migration Parameter (Geschwindigkeit, Wegstrecke, Verdrängung, etc.) quantifiziert mit einem von mehreren Software-Pakete, einschließlich ImageJ, AutoQuant oder Volocity (Abbildung 1). </li><li> Zur Erzeugung eines &quot;Spinnennetzes Grundstück,&quot; die XY-Koordinaten für jede Zelle und Zeitpunkt erhoben werden (Abbildung 2) und auf eine Grafik mit einem gemeinsamen Ausgangspunkt für jede Zelle im Ursprung (Abbildung 3). </li></ol> 5. Repräsentative Ergebnisse Am Tag 6 der Kultur in Anwesenheit von entweder IL-2 oder IL-15, T-LymphozytenMake-up von&gt; 98% der Zellen, wie durch positive CD3-Färbung sowie positive CD4 und / oder CD8-Färbung bestimmt. Für IL-2, fanden wir 83% CD4 +-Zellen, 15% CD8 + und &lt;1% CD4 + CD8 +-Zellen. Für IL-15, fanden wir 88% CD4 +-Zellen, 11% CD8 + und &lt;1% CD4 + CD8 +-Zellen. Während T-Lymphozyten-Migration auf ICAM-1/SDF-1 Substraten zeigten Zellen mit einer Geschwindigkeit von ca. 15 mu m / min, das über eine 1-stündige Frist aufrechterhalten werden kann. T-Lymphozyten-Migration auf ICAM-1/SDF-1 ist abhängig von LFA-1-vermittelte Adhäsion, als Anti-LFA-1-Ligand-blockierenden Antikörper stark hemmt die Migration. <img src="/files/ftp_upload/2017/2017fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Handy-Tracking für die Migration T-Lymphozyten. T-Lymphozyten aus Vollblut isoliert und kultiviert in Gegenwart von IL-2 oder IL-15 für 6 Tage durften halten und auf Glasboden-Schalen mit 10 ug / ml ICAM-1 und beschichtete migrieren 2 pg / mL SDF-1. Die Bilder wurden alle 10 Sekunden für 30 Minuten erworben. Die Zellen wurden in jedem Bild identifiziert und verfolgt im Laufe der Zeit mit Volocity Software. Dieser Film zeigt die zufällige Migration von T-Lymphozyten mit einer Geschwindigkeit von ca. 15 mu m / min. <a href="http://www.jove.com/files/ftp_upload/2017/2017_Kim_Movie1.mov">Klicken Sie hier, um das Video zu sehen.</a> Movie 1. Zelle Verfolgung der wandernden T-Lymphozyten. T-Lymphozyten aus Vollblut isoliert und kultiviert in Gegenwart von IL-2 oder IL-15 für 6 Tage durften halten und auf Glasboden-Schalen mit 10 ug / ml ICAM-1 und 2 pg / mL SDF-1 beschichtet migrieren . Die Bilder wurden alle 10 Sekunden für 30 Minuten erworben. Die Zellen wurden in jedem Bild erkannt und verfolgt im Laufe der Zeit mit Volocity Software. Dieser Film zeigt die zufällige Migration von T-Lymphozyten mit einer Geschwindigkeit von ca. 15 mu m / min. <img src="/files/ftp_upload/2017/2017fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Spatiotemporal Zelle positionieren. Die XY-Koordinaten jeder Zelle wurden für jeden Zeitpunkt mit Volocity Software erhalten. <img src="/files/ftp_upload/2017/2017fig3.jpg" alt="Abbildung 3" /> Abbildung 3. &quot;Spider Web-Plot.&quot; Mit dem xyt Koordinaten für jede Zelle, 15-Zellen nach dem Zufallsprinzip ausgewählt wurden und aufgetragen mit einem gemeinsamen Ausgangspunkt an der Herkunft. Vergleicht man Spinnennetz Parzellen gibt einen schnellen visuellen Darstellung von Unterschieden in der Migration zwischen verschiedenen experimentellen Bedingungen. Grundstücke für T-Lymphozyten-Migration unter Kontrollbedingungen (links) und in Anwesenheit eines anti-LFA-1-Ligand-blockierenden Antikörper (rechts).

<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>

In diesem Experiment, bieten wir Informationen über ein einfaches System, um die Beweglichkeit der primären menschlichen T-Lymphozyten zu analysieren. In-vitro-Assays Migration wurden verwendet, um die Rollen von vielen Molekülen und Signalwege in der Fortbewegung verschiedener Zelltypen beteiligt zu sezieren. Einige kritische Steuerungen im Auge zu behalten, wenn die Gestaltung Ihrer eigenen Experiment aus unserem Protokoll gehören: 1) Non-Klebstoff oder Nicht-Integrin-Haftgrund Beschichtungen, wie Rinders...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<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>
<td>&nbsp;</td>		</tbody>
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human t lymphocyte isolation, culture and analysis of migration in vitro

Die Migration von T-Lymphozyten besteht der Klebstoff Wechselwirkung von Zelloberflächen Integrine mit Liganden auf anderen Zellen oder mit der extrazellulären Matrix exprimiert. Die genaue raumzeitliche Aktivierung der Integrine aus einer geringen Affinität Zustand in einen Zustand hoher Affinität an die Zelle Vorderkante ist für T-Lymphozyten-Migration wichtige<sup&gt; 1</sup&gt;. Ebenso Zurückziehen der Zelle Hinterkante oder uropod, ist ein notwendiger Schritt bei der Aufrechterhaltung persistent Integrin-abhängigen T-Lymphozyten-Motilität<sup&gt; 2</sup&gt;. Viele Therapieansätze für Autoimmun-und entzündlichen Erkrankungen Ziel Integrine als ein Mittel zur Hemmung der übermäßige Rekrutierung und Migration von Leukozyten<sup&gt; 3</sup&gt;. Zur Untersuchung der molekularen Vorgänge, die menschlichen T-Lymphozyten-Migration zu regulieren, haben wir eine genutzt<em&gt; In-vitro-</em&gt; System Zellwanderung auf einer zweidimensionalen Substrat, das die Umgebung, die eine T-Lymphozyten Begegnungen bei der Einstellung aus dem Gefäßsystem imitiert zu analysieren. T-Lymphozyten sind zuerst von menschlichen Spendern isoliert und anschließend angeregt und kultiviert 7-10 Tage. Während des Tests sind T-Lymphozyten erlaubt, festhalten und auf einem Substrat mit ICAM-1 (ICAM-1), einem Liganden für Integrin LFA-1, und Stromazellen cell-derived factor-1 (SDF-1) beschichtet migrieren. Unsere Daten zeigen, dass T-Lymphozyten eine wandernde Geschwindigkeit von ca. 15 mu m / min aufweisen. T-Lymphozyten-Migration kann durch Integrin-Blockade gehemmt werden<sup&gt; 1</sup&gt; Oder durch Inhibitoren des zellulären Actomyosin Maschinen, regelt die Zellwanderung<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

T-Lymphozyten-Migration tritt bei der Referenzfahrt zu lymphatischen Organen, Ausfahrt aus dem Gefäßsystem, und das Eintreten in den peripheren Geweben. Hier beschreiben wir ein Protokoll, das verwendet werden, um T-Lymphozyten-Migration zu analysieren lassen<em&gt; In-vitro-</em&gt;.

Menschliche T-Lymphozyten-Isolierung, Kultur und Analyse der Migration 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 Aufrufe.  University of Rochester. T-Lymphozyten-Migration tritt bei der Referenzfahrt zu lymphatischen Organen, Ausfahrt aus dem Gefäßsystem, und das Eintreten in den peripheren Geweben. Hier beschreiben wir ein Protokoll, das verwendet werden, um T-Lymphozyten-Migration zu analysieren lassen In-vitro-.

Serie: 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.

piggyBac Transposon-System Modifikation von primären menschlichen T-Zellen

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

Forschung

Immunologie und Infektion

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.

Lymphocyte Isolierung von menschlicher Haut für phänotypische Analyse und<em&gt; Ex Vivo</em&gt; Zellkultur

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.

Maus Naive CD4<sup&gt; +</sup&gt; T-Zell-Isolierung und<em&gt; In-vitro-</em&gt; Differenzierung in T-Zell-Populationen

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.

Imaging CD4 T-Zell-Interstitial Migration in den entzündeten 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 ...

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.

Durchflusszytometrische Analyse von natürlichen Killerzellen lytische Aktivität in menschlichem Vollblut

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.

Isolierung und Analyse über Durchflusszytometrie der Menschen Endozervikale Gamma-Delta-T-Zellen

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 von Lymphozyten-Extravasation einer Verwendung<em&gt; In Vitro</em&gt; Modell des Barrier Menschliches Blut-Hirn

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.

Quantifizierung der menschliche Monocyte Chemotaxis In Vitro und murinen Lymphozyten Menschenhandel 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.

Isolation und In-vitro- Kultur von murinen und menschlichen alveoläre Makrophagen

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 der Scherung strömungsinduzierte Migration der Murine Marginal Zone B-Zellen 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, ...