Estamos muy agradecidos a los cirujanos del Departamento de Cirugía Torácica y Cardiovascular, Clínica Universitaria de Tubinga por proporcionarnos las muestras de timo y Bruno Kyewski (DKFZ, Heidelberg, Alemania) para proporcionar el anticuerpo CDR2. También nos gustaría dar las gracias a Hans-Jörg Bühring y Sabrina Grimm de las instalaciones de clasificación (Universidad de Tubinga). Este trabajo fue apoyado por el SFB 685 y la Fundación Hertie.

<ol>
	<li>Miller, J. F. A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+of+thymus+function+and+of+thymus-derived+lymphocytes.">The discovery of thymus function and of thymus-derived lymphocytes.</a> Immunological Reviews. 185, 7-14 (2002).</li><li>Klein, L., Hinterberger, M., Wirnsberger, G., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+presentation+in+the+thymus+for+positive+selection+and+central+tolerance+induction.">Antigen presentation in the thymus for positive selection and central tolerance induction.</a> Nat Rev Immunol. 9, 833-844 (2009).</li><li>Vandenabeele, S., Hochrein, H., Mavaddat, N., Winkel, K., Shortman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+thymus+contains+2+distinct+dendritic+cell+populations.">Human thymus contains 2 distinct dendritic cell populations.</a> Blood. 97, 1733-1741 (2001).</li><li>Gotter, J., Brors, B., Hergenhahn, M., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medullary+Epithelial+Cells+of+the+Human+Thymus+Express+a+Highly+Diverse+Selection+of+Tissue-specific+Genes+Colocalized+in+Chromosomal+Clusters.">Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters.</a> The Journal of Experimental Medicine. 199, 155-166 (2004).</li><li>Rouse, R. V., Bolin, L. M., Bender, J. R., Kyewski, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+reactive+with+subsets+of+mouse+and+human+thymic+epithelial+cells.">Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells.</a> Journal of Histochemistry &amp; Cytochemistry. 36, 1511-1517 (1988).</li><li>Stoeckle, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cathepsin+S+dominates+autoantigen+processing+in+human+thymic+dendritic+cells.">Cathepsin S dominates autoantigen processing in human thymic dendritic cells.</a> Journal of Autoimmunity. 38, 332-343 (2012).</li><li>Woods Ignatoski, K. M., Bingham, E. L., Frome, L. K., Doherty, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+trans-differentiation+of+thymus+cells+into+parathyroid-like+cells+without+genetic+manipulation.">Directed trans-differentiation of thymus cells into parathyroid-like cells without genetic manipulation.</a> Tissue Eng Part C Methods. 17, 1051-1059 (2011).</li><li>Williams, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Cell+Analysis+of+Complex+Thymus+Stromal+Cell+Populations:+Rapid+Thymic+Epithelia+Preparation+Characterizes+Radiation+Injury.">Single Cell Analysis of Complex Thymus Stromal Cell Populations: Rapid Thymic Epithelia Preparation Characterizes Radiation Injury.</a> Clinical and Translational Science. 2, 279-285 (2009).</li><li>Bendriss-Vermare, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+thymus+contains+IFN-&#945;-producing+CD11c-,+myeloid+CD11c+,+and+mature+interdigitating+dendritic+cells.">Human thymus contains IFN-&#945;-producing CD11c-, myeloid CD11c+, and mature interdigitating dendritic cells.</a> The Journal of Clinical Investigation. 107, 835-844 (2001).</li><li>Schmitt, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+characterization+of+human+thymic+dendritic+cell+subsets.">Ex vivo characterization of human thymic dendritic cell subsets.</a> Immunobiology. 212, 167-177 (2007).</li><li>Dzionek, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDCA-4:+Three+Markers+for+Distinct+Subsets+of+Dendritic+Cells+in+Human+Peripheral+Blood.">BDCA-4: Three Markers for Distinct Subsets of Dendritic Cells in Human Peripheral Blood.</a> The Journal of Immunology. 165, 6037-6046 (2000).</li><li>Wu, L., Shortman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+thymic+dendritic+cells.">Heterogeneity of thymic dendritic cells.</a> Seminars in Immunology. 17, 304-312 (2005).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> Journal of Immunological Methods. 385, 23-34 (2012).</li><li>Adamopoulou, E., Tenzer, S., Hillen, N., Klug, P., Rota, I. A., Tietz, S., Gebhardt, M., Stevanovic, S., Schild, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+MHC-peptide+matrix+of+central+tolerance+in+the+human+thymus.">Exploring the MHC-peptide matrix of central tolerance in the human thymus.</a> Nature Communications. , (2013).</li></ol>

Ningún conflicto de interés declarado.

El protocolo aquí descrito es una modificación del protocolo publicado por Gotter et al 4. Los pasos críticos en el protocolo son la condición y la preparación inicial del tejido, así como la separación por densidad de Percoll. Recomendamos para procesar el tejido tan pronto como sea posible después de su recolección. Es importante trabajar rápido pero bien al limpiar y cortar el tejido. Durante el lavado de timocitos se describe en el paso 2.3, es fundamental encontrar el equilibrio adecua...

El timo es el órgano en el que se produce el desarrollo de células T. Su tamaño relativo y absoluto disminuye con la edad cuando se convierte sustituido sucesivamente por la grasa a pesar de la actividad del timo todavía se puede detectar en la vejez. Su importancia para la respuesta inmune se demostró a principios del decenio de 1960 1. El repertorio de células T se forma a través de la interacción de receptores de células T con complejos de péptido-MHC en diferentes tipos de timo de APC, que proporcionan señales de supervivencia o muerte para el desarrollo de las células T, lo que resulta en un repertorio de células T funcionales y en gran medida auto-tolerante 2. Aproximadamente el 98% de las células en el timo humano están desarrollando células T que se hace referencia como timocitos. El 2% restante consiste de un número de diferentes tipos de células, incluyendo una variedad de TEC (cortical, medular, subcapsular), mieloide y plasmocitoide DC (MDC, PDC), macrófagos, células B, células T re-circulantes maduras, granulocitos, Fibroblasts, células endoteliales y células epiteliales muy raras con un fenotipo de expresión se asemeja a la de las células de otros tejidos tales como músculo, neuronas y epitelio respiratorio (Figura 1). De éstos, TEC y DC son los principales tipos de APC se encuentran en un timo normal. En los últimos años, la purificación de estos tipos de APC para la cultura y el perfil molecular ha ganado cada vez más interés. Debido a su baja frecuencia, el aislamiento de cualquiera de estos tipos de células para el análisis detallado requiere un procedimiento eficaz, reproducible y rentable. El método que aquí se presenta es una modificación de los estudios publicados previamente 3,4. Al igual que con cualquier otro tejido, la extracción de células del timo se puede lograr por enzimáticamente la desagregación de la célula-célula y las redes de interacción célula-matriz, con el fin de obtener una suspensión de células individuales. Hay ciertos parámetros como la buena eficiencia de disociación, rendimiento celular, la viabilidad celular y la retención de s célulasmarcadores urface que son cruciales y necesitan ser optimizado para el aislamiento con éxito de estas poblaciones celulares raras. En este protocolo, el aislamiento de la CC y subconjuntos TEC se realiza haciendo una suspensión de una sola célula del tejido por rotura mecánica y digestión enzimática. Utilizamos colagenasa A de Clostridium histolyticum, que tiene una relación equilibrada de las diferentes actividades de la enzima, para romper el colágeno natural que mantiene el tejido juntos. DNasa I se incluye en la solución de la enzima para reducir la agregación celular debido a ADN libre de células muertas (timocitos son muy sensibles). Nosotros también ofrecemos un enfoque alternativo para la digestión enzimática del tejido típico que implica el tratamiento de tejidos mecánica y enzimática asistida por un disociador tejido. La suspensión de células individuales se somete entonces a una sola centrifugación de densidad de Percoll para el enriquecimiento de la fracción de baja densidad (LDF) de las células. A partir de esta fracción de células, DC puede ser aislado por tinción fo marcadores CC-superficie (es decir, CD11c +) y mediante separación magnética o células activadas por fluorescencia (FACS). A diferencia de las células linfoides que comprenden la gran mayoría de las células en el timo, TCE no expresan CD45 en niveles altos, pero son positivos para la molécula de adhesión celular epitelial EpCAM. ctec se puede distinguir de TCE medular por la expresión de un antígeno aún sin definir reconocido por el CDR-2 (dendrítica de reticulocitos-2 cortical) de anticuerpos 4,5 y algo menor expresión de EpCAM. El diferencial de la co-expresión de EpCAM y CDR2 permite el aislamiento eficiente de estos subconjuntos TEC a través de células de alta velocidad de clasificación 6. El protocolo que se presenta aquí está optimizado para el tejido del timo humano. La duración del procedimiento depende de la cantidad de tejido y la capacidad del experimentador, así como la velocidad del clasificador de células, si se utiliza la separación FACS. Normalmente, el protocolo para el aislamiento de CC puede ser completado dentro de 5HR -6 y para el aislamiento de TCE en 8-10 horas. El aislamiento de la CC y subconjuntos TEC a partir de tejido del timo es sensible al tiempo. Cuanto más rápido el procedimiento de aislamiento, mejor la condición de las células. Por último, las células aisladas se pueden utilizar para investigaciones posteriores como estudios comparativos de ARNm y la expresión de proteínas, los experimentos de PCR, aislamiento de las proteínas, el perfil molecular (es decir, transcriptómica, análisis de micro ARN), así como el cultivo de células 6. Declaración de Ética Con el fin de ser capaz de trabajar con el tejido del timo humana que el investigador tiene que obtener la aprobación del comité de ética local o las autoridades responsables, así como un consentimiento informado por escrito del donante (o por lo general sus padres, ya que el tejido se obtiene generalmente de menor de edad los niños). Por otra parte, todos los tejidos humanos deben ser manejados como se deben tomar medidas potencialmente infecciosos y apropiadas, tales como trabajar con guantes, etc.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and Materials</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>PAA</td>
			<td>E15-842</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>PAA</td>
			<td>H15-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum-Gold</td>
			<td>PAA</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>PAA</td>
			<td>K41-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10 103 586 001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I, grade II bovine pancreatic</td>
			<td>Roche</td>
			<td>10 104 159 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 10x in PBS</td>
			<td>PAA</td>
			<td>L11-001</td>
			<td>stock conc. 20 mg/ml</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Protein Labelling kit</td>
			<td>Molecular Probes</td>
			<td>A-10235</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human CDR2 (purified)</td>
			<td>Bruno Kyewski, DKFZ- Heidelberg, Germany</td>
			<td></td>
			<td>labeled with Alexa Fluor 488</td>
		</tr>
		<tr>
			<td>anti-human CD45 (Pacific Blue)</td>
			<td>Biolegend</td>
			<td>304022</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human EpCAM (APC)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-254</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human CD11c (PE)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-411</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PE Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45 Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-045-801</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>for tissue dissociator</td>
		</tr>
		<tr>
			<td>Percoll (density 1.130 g/ml)</td>
			<td>GE Healthcare, Life Sciences</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile distilled Water (DNAse/ RNAse free)</td>
			<td>GIBCO</td>
			<td>10977-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamunex 10%</td>
			<td>Tajecris-Biotherapeutics</td>
			<td>G120052</td>
			<td>1:10 pre-dilution, use 20 &#956;l/1 x 106cells</td>
		</tr>
		<tr>
			<td>0.22 &#956;m filter</td>
			<td>Millex GS</td>
			<td>SLGS033SS</td>
			<td>Syringe driven</td>
		</tr>
		<tr>
			<td>Stericup filter unit</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>Pump driven</td>
		</tr>
		<tr>
			<td>50 ml PC oak ridge centrifuge tubes</td>
			<td>Nalgene</td>
			<td>3118-0050</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>50 ml PP conical tubes</td>
			<td>Becton Dickinson</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm x 75 mm 5 ml test tubes</td>
			<td>Becton Dickinson</td>
			<td>352058</td>
			<td>FACS stainings</td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#956;m</td>
			<td>Becton Dickinson</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>INSTRUMENTS</td>
		</tr>
		<tr>
			<td>Flow Cytometer-Sorter (BD FACSAriaTMIIu)</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Evolution R6 (rotor)</td>
			<td>Kendro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator REAX 2</td>
			<td>Heidolph</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093 235</td>
			<td>tissue dissociator</td>
		</tr>
	</tbody>
</table>

isolation of myeloid dendritic cells and epithelial cells from human thymus

En este protocolo se proporciona un método para aislar células dendríticas (DC) y células epiteliales (TEC) de la timo humano. DC y TEC son la principal célula presentadora de antígeno (APC) tipos que se encuentran en un timo normal y está bien establecido que desempeñan papeles distintos durante la selección tímica. Estas células se localizan en distintos microambientes en el timo y cada tipo de APC constituye sólo una población menor de células. Para entender mejor la biología de estos tipos de células, la caracterización de estas poblaciones de células es altamente deseable, pero debido a su baja frecuencia, el aislamiento de cualquiera de estos tipos de células requiere un procedimiento eficaz y reproducible. Este protocolo detalla un método para obtener células adecuadas para la caracterización de diversas propiedades celulares. Tejido tímico se interrumpe mecánicamente y después de diferentes pasos de la digestión enzimática, la suspensión de células resultante se enriquece usando una etapa de centrifugación de densidad de Percoll. Para el aislamiento de CC mieloide (CD11c &lt;sup&gt; +), las células de la fracción de baja densidad (LDF) se inmunoselecciona por clasificación celular magnética. Enriquecimiento de las poblaciones de TEC (MTEC, ctec) se logra por el agotamiento de las células hematopoyéticas (CD45 hi) de la fracción de células de Percoll de baja densidad que permite su posterior aislamiento a través de células activadas por fluorescencia (FACS) utilizando marcadores de células específicos. Las células aisladas se pueden utilizar para diferentes aplicaciones posteriores.

Como material de partida en este protocolo se utiliza tejido del timo retirado de los niños sometidos a cirugía cardiovascular correctiva (Departamento de Cirugía Torácica y Cardiovascular, Clínica Universitaria de Tubinga) obtenidas después de consentimiento informado y bajo las directrices del centro. Este material de desecho puede variar mucho en tamaño de 2 a 30 g o más. El número de MDC y subconjuntos TEC (ctec y MTEC) que se obtienen dependen del tamaño, así como la edad de la muestra de tejido de timo ...

Watch this Scientific Journal Video about Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus at JoVE.com

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Este protocolo detalla un método para aislar células presentadoras de antígeno de timo humano a través de diferentes etapas de la digestión enzimática del tejido seguido por centrifugación de densidad de la suspensión de células individuales y la clasificación finalmente magnético y / o FACS de las poblaciones de células de interés.

Aislamiento de células dendríticas mieloides y células epiteliales de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

isolation-myeloid-dendritic-cells-epithelial-cells-from-human

Research

JoVE Journal

Immunology and Infection

19.1K vistas.  Hertie Institute for Clinical Brain Research.  Este protocolo detalla un método para aislar células presentadoras de antígeno de timo humano a través de diferentes etapas de la digestión enzimática del tejido seguido por centrifugación de densidad de la suspensión de células individuales y la clasificación finalmente magnético y / o FACS de las poblaciones de células de interés. 

Video: Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Aislamiento de células dendríticas del pulmón de ratón

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Investigación

Inmunología e Infección

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Aislamiento y caracterización de las células dendríticas y los macrófagos del intestino delgado de ratón

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Aislar y inmunoticción Los linfocitos y las células dendríticas de las placas de Peyer murinas de

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

La evaluación de la innata de detección de VIH-1 CD4 infectados<sup&gt; +</sup&gt; Células T por las células dendríticas plasmocitoides Usando un<em&gt; Ex vivo</em&gt; Co-cultura del sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

El aislamiento, la identificación y purificación de células epiteliales del timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Células T Captura bacterias por transinfection de células dendríticas

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generación de células dendríticas derivadas de monocitos humanos de sangre entera

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Aislamiento de las células dendríticas del tracto reproductivo femenino humano estudios fenotípica y funcional

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Desarrollo y validación de una sola molécula ultrasensible matriz Digital análisis de enzima-ligado del inmunosorbente para interferón-α humano

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.