biopsias de piel de pacientes con psoriasis fueron amablemente proporcionados por el Dr. Andreas Koerber (departamento de Dermatología de la Universidad de Essen, Alemania) tras el consentimiento informado por escrito o por vía oral para uso científico. XH también es apoyado por SNCF 61263039 y 11101321 SNCF.

NOTA: Las biopsias de piel de individuos sanos se obtuvieron a partir de restos de piel abdominal de los individuos sometidos a cirugía plástica electiva tras el consentimiento informado por escrito o por vía oral para uso científico. El uso de piel humana fue aprobado y de acuerdo con las normas establecidas por los comités de ética médica para la investigación humana del centro médico de la Universidad Radboud, Nijmegen, Países Bajos y la Universidad de Essen, Alemania. 1. Preparación de suspensiones de células individuales de la piel humana (Trabajo estéril en un gabinete de flujo si el cultivo celular subsiguiente es obligatorio) <ol><li> Preparar medio de cultivo celular: RPMI 1640 + penicilina / estreptomicina (concentraciones finales de 100 unidades / ml y 100 mg / ml, respectivamente) + piruvato (0,02 mM) y glutamax (0,02 mM), sin suero añadido. </li><li> Para preparar el medio de cultivo completo: medio de cultivo preparado en el paso 1.1 + 10% de suero humano agrupado (HPS); almacenar a 4 ° C. Llevar medio a 20 ° C ± 2 frente a nosotrosEn g. </li><li> Obtener la biopsia de la piel utilizando un instrumento de biopsia por punción redonda de 4 mm y mantenerlo en medio de cultivo completo RPMI 1640 a 20 ° C ± 2 para un máximo de 4 horas oa 4 ° C ON. Procesar la biopsia tan pronto como sea posible a su llegada al laboratorio. NOTA: más largo de almacenamiento de la piel influirá en el rendimiento de células y la viabilidad celular. </li><li> Etiquetar un tubo de disociación con tapa azul y añadir 5 ml de medio de cultivo completo en el tubo etiquetado. </li><li> Añadir 2 ml de medio de cultivo completo en cada pocillo (un total de 3 pocillos) de una placa de cultivo estéril de 6 pocillos. Utilice herramientas estériles para colocar la biopsia en un solo pozo, enjuague, moverlo hacia un segundo pozo y repetir este paso una vez más, logrando así un total de tres enjuagues. </li><li> Transferir la biopsia de la piel bien enjuagado a una placa de Petri estéril, añadir 100 l de medio de cultivo completo en la parte superior de la biopsia, y cuidadosamente raspar el tejido graso subcutáneo utilizando un acero inoxidable desechable bisturí estéril. NOTA: Jes que es un paso crítico. </li><li> Cortar cada biopsia de piel en 4 trozos más pequeños en una placa de Petri estéril. Transferir las muestras (hasta cuatro de las biopsias de 4 mm por tubo) en el tubo de disociación preparado que contiene 5 ml de medio de cultivo completo. </li><li> Cerrar herméticamente los tubos con la tapa y coloque boca abajo a la camisa del disociador automatizado de tejidos. Asegúrese de que todo el material de muestra se encuentra en la zona del rotor. </li><li> Iniciar el proceso de disociación mediante la ejecución del &quot;_01 m_spleen programa&quot; (un programa predefinido proporcionada por la memoria interna del instrumento o de la tarjeta de programa acompañada) para disociar la biopsia a la velocidad de rotación apropiada para 56 seg. </li><li> Después del procesamiento, separar el tubo de la disociación del disociador y asegurarse de que todo el material disociado se recoge en la parte inferior del tubo. </li><li> Añadir 150 l de colagenasa IA (80 mg / ml) en el tubo de disociación e incubar la muestra en un baño de agua con agitación a 37° C durante 60 min. Añadir 100 l de DNasa I (5 MU / ml) en tubo de thedissociation, mezclar bien. NOTA: Mayor concentración de colagenasa o el tiempo de incubación más largo va a alterar la viabilidad celular. </li><li> Una el tubo de disociación para la manga de la disociador tejido automatizado y ejecutar el &quot;m_ programa bazo _01&quot; para disociar la biopsia una vez más. </li><li> Colocar un filtro de células de nylon 70 M en la parte superior de un tubo Falcon 50 ml. Aplicar materiales de muestra disociadas a este filtro de células para eliminar grupos de células / restos de tejido. </li><li> Lavar filtro de células una vez con 5 ml de medio de cultivo completo. Centrifugar a 20 ° C ± 2, 450 xg durante 10 min y el sobrenadante aspirado. </li><li> Repita el paso de lavado una vez más. Volver a suspender los sedimentos celulares en 300 l de medio de cultivo completo. una sola célula suspensiones están listos para su posterior análisis; continuar con protocolos para el cultivo de células ex vivo o citometría de flujo análisis. </li><li> En caso de un nuevo itinción de citocinas ntracellular, estimular las células con PMA (12,5 ng / ml), ionomicina (500 ng / ml) y Brefeldine A (5 g / ml) durante 4 horas a 37 ° C, 5% de CO 2 incubadora durante 4 h antes de la realización de flujo El análisis de citometría. </li></ol> 2. policlonal activación de células T cutáneas Residentes (Ex Vivo Cultura de la célula) <ol><li> Alícuota de 100 l suspensiones unicelulares (preparado en la etapa 1.15) en una placa de fondo redondo de 96 pocillos. NOTA: Para cada uno de 4 mm biopsia de la piel, las suspensiones de una sola célula adquiridos se pueden dividir en al menos dos pocillos de una placa de 96 pocillos. </li><li> Añadir anti-CD3 / anti-CD28 microperlas recubiertas con mAb (25.000 cuentas por pocillo), recombinante humana de citoquinas rIL-2 (concentración final 25 U / ml) y rIL-1ß (concentración final 50 ng / ml). </li><li> Cubrir la placa de cultivo con una tapa de placa de marcado, se incuban en 5% de CO 2, el 100% de humedad, 37 ° C incubadora. Cambie medio cuando el color medio se pone en amarillo. <br/&gt; NOTA: Una colonia de células claras se puede observar a partir de día 8 de cultivo celular. </li></ol> 3. Análisis de citometría de flujo de células de la piel primaria / Residentes T cultivadas <ol><li> Preparar tampón FACS: PBS + BSA al 0,2%. </li><li> Transferencia de células de interés en una placa de 96 pocillos de fondo en v. Centrifugar a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Resuspender el precipitado en 100 l de PBS. Centrifugar a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Las células se tiñen con 100 l de eFluorescence780 preparados conjugados colorante de viabilidad corregible (1: 1000 dilución utilizando PBS) a 4 ° C durante 30 min. Añadir 100 l de tampón de FACS, se centrifuga a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Seleccionar mAbs requeridos marcador de superficie celular, por ejemplo: CD45-BV421 (HI30, 01:20), CD3-ECD (UCHT1, 1:50), CD4-PC5.5 (1388,2, 1: 200), CD8-APC-AlexFluo700 (B9.11, 1: 400), CD14-FITC (TUK4, 01:50), CD19-APC-AlexFluo750 (13-119, 1:50), CD56-PE (MEM-188, 1:50), CD25-PeCy7 (FC96, 1:50), CD11c-PeCy7 (BU15, 1:10), y CD1c-APC (AD5-8E7, 01:10), preparar los mAbs mezcla usando tampón de FACS en función de cada factor de dilución mAb probado. Definir ajustes de la compuerta utilizando control de isotipo de anticuerpo junto con la muestra no mancha. </li><li> Añadir 25 l de preparado mAbs-mezcla en cada pocillo. Incubar 20 minutos a 20 ° C ± 2, protegerlo de la luz. </li><li> Añadir 100 l de tampón de FACS, se centrifuga a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Para las muestras que sólo requieren tinción de la superficie celular, continúe con el paso 3.16. </li><li> En el caso de la tinción de Foxp3 intra-celular: <ol><li> Preparar la fijación y permeabilización de amortiguación mediante la mezcla de una parte de concentrado con 3 partes de diluyentes. </li><li> Preparar tampón de permeabilización 1x: 1 parte de tampón de permeabilización 10x + 9 partes de H 2 O. esterilizada </li></ol></li><li> pellets de resuspender en 100 l de fijación y permeabilición tampón, mezclar bien e incubar a 4 ° C durante 30 min. </li><li> Añadir 100 l de tampón de permeabilización a cada pocillo, se centrifuga a 450 xg a TA durante 2 min, y el sobrenadante aspirado. </li><li> Lavar las células con tampón de permeabilización una vez más. </li><li> Seleccionar mAbs intracelulares necesarias, por ejemplo, Foxp3-eFluo450 (PCH101, 01:50), IL-17A-AlexFluo488 (eBio64DEC1, 01:50) y IFN-PeCy7 (4S.B3, 1: 400). Preparar la mezcla de anticuerpos monoclonales usando tampón de permeabilización que contenía suero de rata normal 2% de acuerdo a cada factor de dilución del AcMo probado. Definir ajustes de la compuerta utilizando control de isotipo de anticuerpo junto con la muestra no mancha. </li><li> Añadir 20 l de preparado mAb-mezcla en cada pocillo. Incubar a 4 ° C durante 30 minutos, protegerlo de la luz. </li><li> Después de 30 min de incubación, añadir 100 l de tampón de permeabilización en cada pocillo. Centrifugar a 20 ° C ± 2, 450 xg durante 2 min, y el sobrenadante aspirado. </li><li> Lavar las células con buffe permeabilizaciónr una vez más. sedimento se vuelve a suspender en 110 l de tampón FACS, y las suspensiones de células en un tubo de transferencia de microFACS. NOTA: La muestra está lista para medición mediante citometría de flujo 10-color. </li><li> En el caso del número de células exacta requerida, añadir 10 l de suspensión uniforme bien mezclada de fluoroesferas en cada muestra inmediatamente antes de realizar las mediciones de citometría de flujo. </li></ol>

<ol>
	<li>Clark, R. 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=The+vast+majority+of+CLA++T+cells+are+resident+in+normal+skin.">The vast majority of CLA+ T cells are resident in normal skin.</a> J Immunol. 176, 4431-4439 (2006).</li><li>Zaba, L. C., Fuentes-Duculan, J., Steinman, R. M., Krueger, J. G., Lowes, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+dermis+contains+distinct+populations+of+CD11c+BDCA-1++dendritic+cells+and+CD163+FXIIIA++macrophages.">Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.</a> J Clin Invest. 117, 2517-2525 (2007).</li><li>Gebhardt, T., 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=Memory+T+cells+in+nonlymphoid+tissue+that+provide+enhanced+local+immunity+during+infection+with+herpes+simplex+virus.">Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus.</a> Nature Immunol. 10, 524-530 (2009).</li><li>Boyman, O., 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=Spontaneous+development+of+psoriasis+in+a+new+animal+model+shows+an+essential+role+for+resident+T+cells+and+tumor+necrosis+factor-alpha.">Spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-alpha.</a> J Exp Med. 199, 731-736 (2004).</li><li>Christophers, E., Mrowietz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+infiltrate+in+psoriasis.">The inflammatory infiltrate in psoriasis.</a> Clin Dermatol. 13, 131-135 (1995).</li><li>Jiang, X., 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=Skin+infection+generates+non-migratory+memory+CD8++T(RM)+cells+providing+global+skin+immunity.">Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.</a> Nature. 483, 227-231 (2012).</li><li>Havran, W. L., 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=Limited+diversity+of+T-cell+receptor+gamma-chain+expression+of+murine+Thy-1++dendritic+epidermal+cells+revealed+by+V+gamma+3-specific+monoclonal+antibody.">Limited diversity of T-cell receptor gamma-chain expression of murine Thy-1+ dendritic epidermal cells revealed by V gamma 3-specific monoclonal antibody.</a> Proc Natl Acad Sci U S A. 86, 4185-4189 (1989).</li><li>Campbell, J. J., 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=CCR7+expression+and+memory+T+cell+diversity+in+humans.">CCR7 expression and memory T cell diversity in humans.</a> J Immunol. 166, 877-884 (2001).</li><li>He, X., 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=Targeting+PKC+in+human+T+cells+using+sotrastaurin+(AEB071)+preserves+regulatory+T+cells+and+prevents+IL-17+production.">Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production.</a> J Invest Dermatol. 134, 975-983 (2014).</li><li>Clark, R. 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=A+novel+method+for+the+isolation+of+skin+resident+T+cells+from+normal+and+diseased+human+skin.">A novel method for the isolation of skin resident T cells from normal and diseased human skin.</a> J Invest Dermatol. 126, 1059-1070 (2006).</li><li>Clark, R. A., Kupper, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+and+dermal+fibroblasts+induce+proliferation+of+natural+regulatory+T+cells+isolated+from+human+skin.">IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin.</a> Blood. 109, 194-202 (2007).</li><li>Keijsers, R. R., 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=In+vivo+induction+of+cutaneous+inflammation+results+in+the+accumulation+of+extracellular+trap-forming+neutrophils+expressing+RORgammat+and+IL-17.">In vivo induction of cutaneous inflammation results in the accumulation of extracellular trap-forming neutrophils expressing RORgammat and IL-17.</a> J Invest Dermatol. 134, 1276-1284 (2014).</li><li>Hybbinette, S., Bostrom, M., Lindberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+dissociation+of+keratinocytes+from+human+skin+biopsies+for+in+vitro+cell+propagation.">Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation.</a> Experimental dermatology. 8, 30-38 (1999).</li><li>Hennino, 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=Skin-infiltrating+CD8++T+cells+initiate+atopic+dermatitis+lesions.">Skin-infiltrating CD8+ T cells initiate atopic dermatitis lesions.</a> J Immunol. 178, 5571-5577 (2007).</li></ol>

The authors have nothing to disclose.

A continuación, se presenta un protocolo para aislar eficazmente las células T residentes de la piel a partir de biopsias de piel humana. La ventaja de este protocolo es el aislamiento de un número relativamente alto de linfocitos viables, y la expresión de marcadores de superficie correspondientes. Los subconjuntos de células identificadas fueron:, células CD4 + y linfocitos T CD8 + y CD25 + Foxp3 + CD11c + DCS. Es importante destacar que la cultura ex vi...

La piel, como la interfaz principal entre el cuerpo y el medio ambiente, proporciona la primera línea de defensa contra la física externa, químicas y biológicas insultos tales como heridas, la radiación ultravioleta y los microorganismos. Piel comprende dos compartimientos principales, la epidermis y la dermis, y contiene una variedad de células inmunes incluyendo las células de Langerhans, macrófagos, células dendríticas (DC), y cerca de 20 millones de células T de memoria, casi dos veces el número presente en todo el volumen de sangre 1 , 2. Una creciente cantidad de datos apoyan la noción de que la piel tiene funciones inmunológicas esenciales, tanto durante la homeostasis del tejido y en diversas condiciones patológicas. Se cree que las células inmunes residentes en la piel normal para llevar a cabo la inmunovigilancia 3 y se ha demostrado que desempeñan un papel en el desarrollo de trastornos inflamatorios tales como psoriasis 4. En la piel lesional psoriásica, tanto CD4 + y CD8 + se infiltraron las células T eran observed y se demostró que la relación de la CD4 y CD8 varía en función del estado de la enfermedad 5. Sin embargo, estas poblaciones de células son difíciles de estudiar porque las técnicas actuales permiten el aislamiento de sólo unas pocas células. Las técnicas actualmente ampliamente utilizados para el aislamiento de células T de la piel humana se combinan disociación mecánica de la piel con tratamiento enzimático. Las biopsias de piel humana son ampliamente picada y se incubaron con enzimas como la tripsina, colagenasa y / o EDTA 6-8. Teniendo en cuenta que la piel es un tejido de barrera que es altamente resistente a las fuerzas de tracción y desagregación mecánica, los métodos establecidos de aislamiento de células T producen muy pocas células, e incluso un menor número de células viables, lo que hace ex vivo de cultivo de células de estas poblaciones de células difícil y desafiante. Aquí, se presenta un método modificado para aislar los linfocitos de la piel humana psoriásica saludable y participar mediante la combinación de mechandisociación ical de la piel usando un disociador de tejido automatizado en lugar del método establecido de picado ampliamente, junto con la digestión enzimática usando colagenasa. Se observaron varios subconjuntos de células inmunes viables, incluyendo países en desarrollo y las células T después de la preparación de una suspensión de una sola célula. Es importante destacar que la expresión de los marcadores de superficie CD3, CD4 y CD8 se conservó bien. Las células preparadas de este modo, están listos para su uso en cultivos de células ex vivo o análisis de citometría de flujo. Este protocolo se ha empleado con éxito para el análisis de biopsias de piel individuales (4 mm) derivadas de la piel lesional de pacientes con psoriasis. Los resultados mostraron que las células T de la piel del paciente residente producen citoquinas inflamatorias como la más IL-17 y IFN en comparación con voluntarios sanos 9.

<table border="0" cellpadding="0" cellspacing="0" width="843">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Name of Reagent/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:299px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch</td>
			<td>Kai Europe</td>
			<td>BP-40F</td>
			<td>4 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable sterile scalpels</td>
			<td>Dalhausen</td>
			<td>1100000510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS C tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td>Blue-capped, used as the dissociation tube.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">gentleMACS Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td style="width:299px;">Automated tissue dissociator. Using the &#34;program spleen_01&#34; for dissociation of the skin biopsy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer&#160;</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">352350</td>
			<td>70 &#181;m nylon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">96-well U-bottom plate</td>
			<td style="width:145px;">Greiner Bio-One</td>
			<td style="width:119px;">650180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>22409-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">GlutaMAX</td>
			<td>Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:281px;">Human Pooled Serum (HPS)</td>
			<td style="width:145px;">in house prepared</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Collagenase I</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C2674</td>
			<td>Type 1-A, suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">DNase I</td>
			<td style="width:145px;">Calbiochem</td>
			<td style="width:119px;">260913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PBS</td>
			<td style="width:145px;">B Braun</td>
			<td style="width:119px;">3623140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BSA</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">A4503-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fixable Viability Dye (FVD) APC-eFluo780</td>
			<td>eBioscience</td>
			<td>65-0865-18</td>
			<td style="width:299px;">Stain dead cells prior to cell fixation; dilute with PBS at 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Concentrate</td>
			<td>eBioscience</td>
			<td>00-5123-43</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Diluent</td>
			<td>eBioscience</td>
			<td>00-5223-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permeabilization Buffer (10x)</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BV421 Mouse anti-human CD45</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">563879</td>
			<td>Clone: HI30; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">FITC Mouse anti-human CD14</td>
			<td>Dako</td>
			<td style="width:119px;">T0844</td>
			<td>Clone: TUK4; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PE Mouse anti-human CD56</td>
			<td style="width:145px;">Dako</td>
			<td style="width:119px;">R7127</td>
			<td>Clone: MOC-1; dilution factor 1:50</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:281px;">ECD Mouse anti-human CD3</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A07748</td>
			<td style="width:299px;">Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PC5.5 Mouse anti-human CD4</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">B16491</td>
			<td>Clone: 13B8.2; dilution factor 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD11c</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A80249</td>
			<td>Clone: BU15; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC Mouse anti-human CD1c</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-090-903</td>
			<td>Clone: AD5-8E7; dilution factor 1:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC-AlexFluo700 Mouse anti-human CD8</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A66332</td>
			<td>Clone: B9.11; dilution factor 1:400</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:281px;">APC-AlexFluo750 Mouse anti-human CD19</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A94681</td>
			<td>Clone: J3-119; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD25</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">AD5-8E7</td>
			<td>Clone: BC96; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">&#160;PE Rat anti-human CLA</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-091-635</td>
			<td>clone: HECA-452; dilution factor 1:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">eFluo450 Rat anti-human Foxp3</td>
			<td>eBIoscience</td>
			<td>48-4776-42</td>
			<td style="width:299px;">Clone: PCH101; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">AlexFluo488 Mouse anti-human IL-17A</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">53-7179-42</td>
			<td style="width:299px;">Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">PeCy7 Mouse anti-human IFNg</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">25-7319-41</td>
			<td style="width:299px;">Clone: 4S.B3; intracellular staining; dilution factor 1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Flow-Count Fluorospheres</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td align="right" style="width:119px;">7547053</td>
			<td>Counting beads, for flow cytometry</td>
		</tr>
	</tbody>
</table>

lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture

La piel humana tiene una función de barrera importante y contiene diversas células inmunes que contribuyen a la homeostasis del tejido y la protección contra los agentes patógenos. A medida que la piel es relativamente fácil acceso, que proporciona una plataforma ideal para estudiar los mecanismos de regulación inmune periférica. células residentes inmunes en sano conducta piel inmunovigilancia, pero también juegan un papel importante en el desarrollo de trastornos inflamatorios de la piel, tales como psoriasis. A pesar de ideas emergentes, nuestra comprensión de la biología subyacente de diversas enfermedades inflamatorias de la piel es aún limitada. Hay una necesidad de una buena calidad de las poblaciones (individual) de células aisladas a partir de muestras de piel biopsia. Hasta ahora, los procedimientos de aislamiento han sido seriamente obstaculizados por la falta de la obtención de un número suficiente de células viables. El aislamiento y el análisis posterior también se han visto afectados por la pérdida de marcadores de linaje de células inmunes, debido a la tensión mecánica y química causada por los procedimientos de disociación actuales para obtenersuspensión de células individuales. A continuación, describimos un método modificado para aislar las células T de la piel humana psoriásica saludable y participar mediante la combinación de disociación mecánica de la piel utilizando un disociador automatizado de tejidos y el tratamiento de colagenasa. Esta metodología conserva la expresión de la mayoría de los marcadores de linaje inmunes tales como CD4, CD8, Foxp3 y CD11c en la preparación de suspensiones de células individuales. Se muestran ejemplos de éxito de aislamiento de células T CD4 + y la posterior fenotípica y análisis funcional.

El protocolo que aquí se presenta producirá entre 2.200 ± 615 (media ± SEM, piel de voluntarios sanos) hasta 178.000 ± 760 (media ± SEM, piel lesionada de pacientes con psoriasis) linfocitos viables de la piel humana cuando se utiliza una sola biopsia de piel de 4 mm. Se identificaron diferentes tipos de células CD45 + en una sola célula suspensiones derivadas de la piel de individuos sanos, incluyendo las células T CD4 + (~ 45%), las células T CD8 +</sup...

Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

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 ...

lymphocyte-isolation-from-human-skin-for-phenotypic-analysis-ex-vivo

Research

JoVE Journal

Immunology and Infection

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

14.4K vistas.  Radboud University Medical Centre. 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.

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

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

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

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

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

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 ...

Investigación

Inmunología e Infección

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA-Ig celdas en función de antígeno artificiales presentación para mejorar la eficiencia Ex vivo Ampliación de los Derechos Humanos CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

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 ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

Preparación de suspensiones de una sola célula para el Análisis cytofluorimetric de diferentes regiones de piel de ratón

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Aislamiento y<em&gt; Ex Vivo</em&gt; Cultura de Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Las células γδ T, una célula αβT Progenitor Extrathymic

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Aislamiento de leucocitos infiltrantes de ratón Piel Usando enzimática Digest y Separación de degradado

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murina de linfocitos Etiquetado por<sup&gt; 64</sup&gt; Cu-Anticuerpo objetivo de receptores de<em&gt; En Vivo</em&gt; Tráfico de células mediante PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine 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 ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

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

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

Evaluación de la migración de linfocitos en un sistema de transmigración ex Vivo

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

El aislamiento de linfocitos de la piel humana para el Análisis y fenotípica

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Preparación de suspensiones de una sola célula para el Análisis cytofluorimetric de diferentes regiones de piel de ratón

Aislamiento y

Aislamiento de leucocitos infiltrantes de ratón Piel Usando enzimática Digest y Separación de degradado

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Murina de linfocitos Etiquetado por

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

Evaluación de la migración de linfocitos en un sistema de transmigración ex Vivo