Este proyecto fue financiado por los Institutos Nacionales de Salud Subvenciones HL087088 (MK) y HL18208 (MK).

1. Aislamiento de linfocitos T humanos <ol><li> Obtener sangre humana de un donante sano. Permitir que la sangre se enfríe a temperatura ambiente (~ 30 min) antes de proceder al siguiente paso. </li><li> Suavemente la pipeta de 3 ml de la temperatura ambiente media del gradiente de densidad Polimorfia en un tubo de poliestireno de 8 ml de fondo redondo. Suavemente agregar 3 ml de sangre total en la parte superior de los medios de comunicación Polimorfia. Es importante evitar la mezcla de los dos reactivos. </li><li> Centrifugar los tubos a 500 xg durante 45 minutos a temperatura ambiente. </li><li> Después de la centrifugación, las células mononucleares de sangre periférica (CMSP) se han separado de otros componentes sanguíneos en la capa de células superior. La capa de PBMC parece, de arriba hacia abajo, como la banda de nubes en primer lugar. </li><li> Retire con cuidado las claras de color amarillo fase superior de la sangre, por encima de la capa de PBMC, y luego usar una micropipeta P1000 para la transferencia de la capa de PBMC a una de 15 ml o 50 ml de tubo cónico. </li><li> Lave la PBMC dos veces con PBS, centrifugar las células a 500 xg durante 5 minutos cada vez. El sobrenadante será algo nublado después de cada lavado. </li></ol> 2. Cultura de los linfocitos T humanos <ol><li> Con una pipeta, transferir los PBMC de un frasco T-75 en 20 ml de la cultura medio RPMI 1640 con 10% de SFB, 1% penicilina / estreptomicina, y 1 mg / ml fitohemaglutinina (PHA). </li><li> Se incuba a 37 ° C y 5% de CO 2 por lo menos durante una hora, y hasta 24 horas. Este paso permite a los monocitos, que se adhiere a la superficie de frasco, a separarse de los linfocitos que permanecen en suspensión. Si una incubación corta (1 hora) se utiliza en este paso, es aceptable el uso de medio RPMI 1640 con 10% de SFB y 1% de penicilina / estreptomicina sin complementar con la APS como se especifica en el paso 2.1. </li><li> Retire con cuidado todos los medios de comunicación desde el matraz, añadir a un tubo cónico de 50 ml y centrifugar a 500 xg durante 5 minutos. </li><li> Resuspender el botón celular, que ahora contiene principalmente los linfocitos, las células y la transferencia a un nuevo T-75 frasco contiene 25 ml de RPMI 1640 con 10% de SFB, 1% penicilina / estreptomicina, y 1 mg / ml de PHA. </li><li> Se incuba a 37 ° C durante 3 días (2 días si la primera incubación de PBMC fue durante la noche). Después de 24 horas de crecimiento, puede ser necesario añadir 15-20 ml de medio fresco y la transferencia a un T-175 más grandes matraz. </li><li> Después de 3 días, utilizar una pipeta para retirar los medios de comunicación y los linfocitos suspendido del matraz y un tubo cónico de 50 ml. Centrifugar a 500 xg durante 5 minutos. </li><li> Resuspender el botón celular y las células de la transferencia a un nuevo frasco T-75 o T-175 que contiene 25 ml (T-75) o 50 ml (T-175) RPMI 1640 con 10% de SFB, 1% penicilina / estreptomicina, y 20 ng / mL de IL-2 o IL-15. </li><li> Linfocitos crecen de 4-7 días. Si se comienza con un frasco T-75, la cultura tendrá que ser ampliado y trasladado a un matraz T-175 después de 1-2 días. </li></ol> 3. En el ensayo in vitro de la migración de linfocitos <ol><li> 1 día antes de la prueba de la migración, un abrigo con fondo de vidrio 0,17 mm plato con 20 mg / ml de proteína A o G en PBS durante una noche a 4 ° C. </li><li> Lavar el plato extensamente con PBS. </li><li> Inmovilizar humanos ICAM-1/Fc (10 mg / mL) y humanos SDF-1 (2 mg / mL) mediante la adición de estos reactivos en una solución de PBS para el plato y la incubación de 4 horas a temperatura ambiente. </li><li> Prepare los linfocitos T, en primer lugar la determinación de la densidad de las células en cultivo en el matraz T-175 con un hemocitómetro. Aproximadamente 2.5 x 10 5 células se debe utilizar por plato para conseguir una densidad celular apropiada para el análisis de la migración. </li><li> Lave los linfocitos T dos veces con PBS y luego volver a suspender en 1 ml de L-15 los medios de comunicación que contiene 1 mg / ml de D-glucosa. </li><li> Lavar el plato cubierto con ICAM-1/Fc y SDF-1 extensamente con PBS. </li><li> La transferencia de linfocitos T en un ml de medio para el plato y mantenerlos a 37 ° C. </li></ol> 4. Captura de una secuencia de imágenes Uso de NIS Software Elementos <ol><li> Abrir NIS software Elements. </li><li> En el menú de ajustes de la cámara, seleccione &quot;binning 2x2&quot; como el modo de dos imágenes en vivo y captura de imágenes. </li><li> Vaya al menú Aplicaciones y seleccione Definir / experimento se ejecute. </li><li> Elija la cantidad de tiempo entre las imágenes y la longitud total de la secuencia de captura de imágenes. </li><li> Pulse el botón &quot;Ejecutar&quot; para iniciar la adquisición de imágenes. </li><li> Las células se pueden seguir y los parámetros de la migración (velocidad, longitud de la trayectoria, desplazamiento, etc) cuantificó usando uno de varios paquetes de software, incluyendo ImageJ, AutoQuant o Volocity (Figura 1). </li><li> Para generar un &quot;complot tela de araña&quot;, las coordenadas xy para cada celda y punto de tiempo se recogen (Figura 2) y se proyecta sobre un gráfico con un punto de partida común para cada celda en el origen (Figura 3). </li></ol> 5. Resultados representante El día 6 de cultivo en presencia de IL-2 o IL-15, los linfocitos Tconstituyen&gt; 98% de las células, según lo determinado por tinción CD3 positivos, así como CD4 positivos y tinción / o CD8. De IL-2, se encontró 83% de CD4 + en las células, el 15% CD8 + y &lt;1% de CD4 + CD8 +. De IL-15, encontramos 88% de CD4 + en las células, el 11% CD8 + y &lt;1% de CD4 + CD8 +. Durante la migración de los linfocitos T en ICAM-1/SDF-1 sustratos, las células mostraron una velocidad de aproximadamente 15 micras / min que puede ser mantenido durante un período de 1 hora. La migración de los linfocitos T en ICAM-1/SDF-1 depende de LFA-1 mediada por la adhesión, como un anti-LFA-1 ligando anticuerpo bloqueante inhibe seriamente la migración. <img src="/files/ftp_upload/2017/2017fig1.jpg" alt="Figura 1" /> Figura 1. Seguimiento de la migración de células linfocitos T. Linfocitos T aislados de sangre total y se cultivan en presencia de IL-2 o IL-15 durante 6 días se permite que se adhieran y migrar a fondo de cristal platos recubiertos con 10 mg / mL de ICAM-1 y 2 mg / mL SDF-1. Las imágenes fueron adquiridas cada 10 segundos durante 30 minutos. Las células fueron identificadas en cada imagen y el seguimiento en el tiempo utilizando el software Volocity. Esta película muestra la migración al azar de los linfocitos T a una velocidad de unos 15 m / min. <a href="http://www.jove.com/files/ftp_upload/2017/2017_Kim_Movie1.mov">Haga clic aquí para ver el video.</a> Movie 1. Celular de seguimiento de la migración de los linfocitos T. Los linfocitos T aislados de sangre total y se cultivan en presencia de IL-2 o IL-15 durante 6 días se permite que se adhieran y migrar en los platos con fondo de vidrio recubierta con 10 mg / mL de ICAM-1 y 2 mg / ml SDF-1 . Las imágenes fueron adquiridas cada 10 segundos durante 30 minutos. Las células fueron identificadas en cada imagen y el seguimiento en el tiempo utilizando el software Volocity. Esta película muestra la migración al azar de los linfocitos T a una velocidad de unos 15 m / min. <img src="/files/ftp_upload/2017/2017fig2.jpg" alt="Figura 2" /> Figura 2. Posición de la célula espacio-temporal. Las coordenadas XY de cada célula se obtuvieron para cada momento el uso de software Volocity. <img src="/files/ftp_upload/2017/2017fig3.jpg" alt="Figura 3" /> Figura 3. &quot;Spider parcela web.&quot; Utilizando el XYT coordenadas para cada celda, 15 células fueron escogidos al azar y se representa con un punto de partida común en el origen. Comparando las parcelas tela de araña da una representación visual rápida de las diferencias en la migración entre diferentes condiciones experimentales. Parcelas para la migración de los linfocitos T en condiciones control (izquierda) y en presencia de un anti-LFA-1 ligando anticuerpo bloqueante (panel derecho).

<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>

En este experimento, se proporcionan detalles sobre un sistema sencillo para analizar la movilidad de primaria linfocitos T humanos. En los ensayos de migración in vitro se han utilizado para analizar el papel de muchas moléculas y vías de señalización implicadas en la locomoción de los diversos tipos de células. Algunos controles críticos a tener en cuenta a la hora de diseñar su propio experimento de nuestro protocolo son: 1) revestimientos adhesivos no adhesivo o no la integrina-su...

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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>
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<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 migración de los linfocitos T implica la interacción adhesiva de las integrinas de la superficie celular con ligandos expresados ​​en otras células o con proteínas de la matriz extracelular. La activación precisa espacio-temporal de las integrinas de un estado de baja afinidad a un estado de alta afinidad en el borde de la célula principal es importante para la migración de los linfocitos T<sup&gt; 1</sup&gt;. Del mismo modo, la retracción del borde de la célula de seguimiento o uropod, es un paso necesario en el mantenimiento persistente de la integrina dependientes de la motilidad de linfocitos T<sup&gt; 2</sup&gt;. Muchos enfoques terapéuticos para las integrinas enfermedades autoinmunes o inflamatorias objetivo como un medio para inhibir la contratación excesiva y la migración de los leucocitos<sup&gt; 3</sup&gt;. Para estudiar los eventos moleculares que regulan la migración de linfocitos T humanos, hemos utilizado un<em&gt; In vitro</em&gt; Sistema para analizar la migración de células sobre un sustrato de dos dimensiones que simula el medio ambiente que un linfocito T se encuentra en la contratación de la vasculatura. Los linfocitos T son los primeros aislados de donantes humanos y luego se estimula y se cultivan durante siete a diez días. Durante el ensayo, los linfocitos T se permite que se adhieran y migrar en un sustrato recubierto con una molécula de adhesión intercelular-1 (ICAM-1), un ligando de la integrina LFA-1, y derivado de células estromales factor-1 (SDF-1). Nuestros datos muestran que los linfocitos T presentan una velocidad migratoria de unos 15 m / min. La migración de los linfocitos T pueden ser inhibidas por bloqueo de la integrina<sup&gt; 1</sup&gt; O por los inhibidores de la maquinaria celular que regula la actomiosina la migración celular<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 migración de los linfocitos T se produce durante la recalada de los órganos linfoides, la salida de la vasculatura, y entrar en los tejidos periféricos. Aquí se describe un protocolo que se puede utilizar para analizar la migración de los linfocitos T<em&gt; In vitro</em&gt;.

Aislamiento de linfocitos T humanos, cultura y análisis de la migración 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 vistas.  University of Rochester. La migración de los linfocitos T se produce durante la recalada de los órganos linfoides, la salida de la vasculatura, y entrar en los tejidos periféricos. Aquí se describe un protocolo que se puede utilizar para analizar la migración de los linfocitos T In vitro.

Video: 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 Transposón modificación del sistema de células T humanas primarias

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

Investigación

Inmunología e Infección

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.

El aislamiento de linfocitos de la piel humana para el Análisis y fenotípica<em&gt; Ex Vivo</em&gt; Cultivo Celular

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.

Ratón Naïve CD4<sup&gt; +</sup&gt; Aislamiento de células T y<em&gt; In vitro</em&gt; Diferenciación en subconjuntos de células 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.

La migración de células T CD4 obtener imágenes intersticial en la dermis inflamadas

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.

Análisis de citometría de flujo de las células asesinas naturales lítico Actividad en sangre entera humana

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.

Aislamiento y Análisis de citometría de flujo, de 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 ...

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.

Análisis de linfocitos extravasación El uso de una<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro humano

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.

Cuantificación de la Quimiotaxis de monocitos humanos In Vitro y linfocitos murinos, tráfico en 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.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y humanos

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.

Análisis de cortante inducida por el flujo de migración de las células B de zona Marginal de Murine 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, ...