Este trabajo fue financiado por los Institutos Nacionales de Salud (NIH), concede R01-DK080752 y P50-DK064540. Damos las gracias a J. Loughman por sus esfuerzos en el establecimiento de este ensayo.

1. El cultivo de 5.637 células de la vejiga epiteliales Realice los siguientes pasos en una campana de cultivo de tejidos de flujo laminar preparado con la irradiación UV y limpiada con etanol al 70%. <ol><li> Prepare medio RPMI-1640 que contenía suero bovino fetal al 10% (FBS) [denominado RPMI +], esterilizar por filtración usando un filtro de 0,22 micras de tamaño de poro, y se calienta a 37 ° C en un baño de agua. </li><li> Descongelar un criovial de 5.637 células (American Type Culture Collection HTB-9; derivada de carcinoma de vejiga) en un 37 ° C baño de agua. Transfiera rápidamente las células descongeladas a un matraz de cultivo tisular de 75 cm2 contiene 20 ml de RPMI +. </li><li> Incubar el matraz a 37 ° C en una atmósfera humidificada con 5% de CO 2 hasta que las células son de aproximadamente 95% de confluencia (~ 6 x 10 6 células por matraz), alrededor de 4 días. </li><li> Para subcultivar las células 5637: <ol><li> Retire el medio de la petaca. Lavar las células con 10 ml de Dulbecco tamponada con fosfato Saline (DPBS) a temperatura ambiente. </li><li> Añadir 6 ml de agua tibia (37 ° C) 0,05% de tripsina, solución de EDTA 0,02% al matraz, y se incuba a 37 ° C con 5% de CO 2 durante 15 min. </li><li> Transferir las células a un tubo cónico de 15 ml, y sedimento por centrifugación a 300 xg durante 5 min. Retire la solución de tripsina / EDTA. </li><li> Resuspender las células en 6 ml de RPMI + (10 6 células / ml), y de transferencia de 1 ml (10 6 células) a un nuevo matraz de 75 cm 2 que contiene 20 ml de RPMI +. </li></ol></li><li> Repita el paso 1.4 subcultivar células cada 4 días o cuando las células alcanzan el 95% de confluencia. </li></ol> 2. Sembrando y Creciendo 5.637 celdas en soportes permeables Realice los siguientes pasos en una campana de cultivo de tejidos utilizando una técnica aséptica. Para obtener resultados óptimos, utilice 5.637 células que han sufrido menos de 10 subculturas. <ol><li> Usando una pinza estéril, invertir los soportes permeables en un 25 mm placa de cultivo de tejido profundo estéril. </li><li&gt; trypsinize un 75 cm 2 frasco de 5.637 células a 95% de confluencia de acuerdo a los pasos 1.4.1-1.4.3. </li><li> Usando una pipeta de 1000 l, resuspender suavemente las células en ~ 2 ml de RPMI + a una concentración de 3 x 10 6 células / ml, determinado por recuento de células usando un hemocitómetro. </li><li> Aplicar 50 l de la suspensión celular (1,5 x 10 5 células) a cada soporte permeable sin tocar la membrana. Coloque la tapa en el plato y coloque cuidadosamente el plato a 37 ° C con 5% de CO2 durante no más de 16 horas. </li><li> Usando una pinza estéril, a la derecha los soportes permeables en una placa de 24 pocillos que contenía 0,6 ml de RPMI + por pocillo. Añadir 0,1 ml de RPMI + para el depósito superior de cada soporte permeable. Incubar a 37 ° C con 5% de CO 2. </li><li> Sustituya el medio cada 2 días. En primer lugar, el medio aspirado desde el depósito superior seguido por el depósito inferior, y luego se aplica fresco RPMI + en el orden inverso, para el depósito inferior (0,6 ml) y después la parte superior reservoir (0,1 ml). </li><li> Siete días después de la siembra de los soportes permeables con 5637 células, evaluar confluencia de las células. Llene el depósito superior con RPMI + (~ 0,35 ml). Las células están suficientemente confluentes cuando el medio no se equilibre entre los depósitos superior e inferior. </li></ol> 3. Preparación del inóculo bacteriano <ol><li> Utilizando una técnica aséptica, añadir 20 ml de caldo de cultivo apropiado a un matraz de 250 ml con una tapa. Utilice Luria-Bertani (LB) caldo para E. coli culturas y añadir antibióticos al caldo en su caso. </li><li> El uso de un asa de siembra estéril, inocular el caldo con las bacterias de la madre de glicerol o una placa de racha. Para las cepas UPEC, incubar el cultivo de bacterias a 37 ° C durante aproximadamente 16 horas, sin agitación (para promover la producción de pili tipo 1). </li><li> Transferir el cultivo bacteriano a un tubo de centrífuga. Sedimenten las bacterias por centrifugación a 8000 xg durante 10 min. </li><li> Decantar el sobrenadante, Y resuspender las bacterias en PBS a OD 600 = 1,000, equivalente a ~ 10 9 UFC / ml. <ol><li> Para generar un estímulo bacteriana muerta por calor, incubar una alícuota (por ejemplo, ~ 1,5 x 10 8 UFC en 150 l) de las bacterias se resuspendieron a 55 ° C durante 30 min. Placa de una alícuota de la suspensión de calor mató a confirmar la muerte bacteriana. </li></ol></li><li> Inmediatamente antes de su uso, diluir las bacterias resuspendidas (vivos o muertos-calor) de 10 veces a 10 8 UFC / ml en caliente sin suero RPMI [denominado RPMI-] en un tubo de microcentrífuga. </li></ol> 4. Aislamiento de los neutrófilos humanos de sangre periférica La recolección de sangre de voluntarios adultos requiere la revisión y la aprobación de una junta de revisión institucional de antelación. Use el equipo de protección personal apropiado y disponer adecuadamente de materiales peligrosos para evitar la exposición a la sangre humana. <ol><li> Aproximadamente 25 ml de sangre venosa de un adulto sano vVOLUNTARIO debe arrastrar a 3 tubos de recogida de sangre que contiene heparina de sodio estéril por el personal capacitado en la flebotomía. <ol><li> Envuelva un torniquete de goma alrededor de la parte superior del brazo, por encima del codo. </li><li> Desinfectar el sitio de entrada, la fosa antecubital, utilizando un 70% de alcohol estéril limpiar. </li><li> Fije un soporte de tubo de plástico a un sistema de agujas de alas de mariposa. </li><li> Inserte la aguja en una vena antecubital en un ángulo de 30 º o menos, bisel hacia arriba. La aguja ha perforado la vena cuando un chorro de sangre aparece en el tubo de plástico. </li><li> Insertar un tubo de recogida de sangre en el soporte de tubo de plástico. Cuando el primer tubo de recogida está lleno, cámbielo por otro del segundo tubo de recogida. Repita con el tercer tubo. </li><li> Cuando el tercer tubo de recogida es de aproximadamente la mitad, retire el torniquete. </li><li> Cubra el sitio de punción con un algodón estéril y retire lentamente la aguja de la vena. Deslice el escudo protector sobre la aguja y el lugar en el quena de riesgo biológico Envase de los sostenidos. </li><li> Aplique presión sobre la zona de punción. Cubra la bola sitio de la punción y el algodón con un vendaje adhesivo. </li><li> Invierta suavemente los tubos de recogida de sangre para dispersar a la heparina. </li></ol></li></ol> Realice los siguientes pasos en una campana de cultivo de tejidos utilizando una técnica aséptica. <ol start="2"><li> Usando una pipeta de 10 ml, transferir suavemente la sangre a un tubo cónico de 50 ml estériles fresco. Añadir un volumen equivalente de 3% (W / V) de dextrano en NaCl al 0,9% y se mezcla por inversión. Incubar el tubo en posición vertical a temperatura ambiente durante 20 min. </li><li> Sin perturbar la capa inferior, aspirar cuidadosamente la capa superior y la transfiere a un nuevo tubo cónico de 50 ml. Sedimentar las células por centrifugación a 300 xg durante 10 min. Eliminar el sobrenadante. </li><li> Resuspender el sedimento celular en un volumen de 0,9% de NaCl equivalente al volumen de partida de la sangre. </li><li> Capa 10 ml de solución de centrifugación de densidad en virtud de la suspensión de células, pla reserva de la interfaz entre las dos fases. Centrifugar a 400 xg durante 30 min sin freno. Eliminar el sobrenadante. </li><li> Para lisar las células rojas de la sangre restantes, resuspender el sedimento celular en 10 ml de frío 0,2% de NaCl. Incubar durante 30 segundos, y luego añadir rápidamente 10 ml de frío 1,6% de NaCl para restablecer la isotonicidad. </li><li> Sedimentar las células por centrifugación a 300 xg durante 6 min. Eliminar el sobrenadante. </li><li> Repetir los pasos 4.6 a 4.7 hasta que el sedimento celular parece estar libre de células rojas de la sangre, típicamente 3 rondas de lisis. </li><li> Usando una pipeta de 1000 l, resuspender el sedimento celular, principalmente de PMN, en caliente (37 ° C) RPMI-a una concentración de 10 7 células / ml, determinada por recuento de células usando un hemocitómetro. Mantener las células a 37 ° C hasta su uso. Típicamente, la viabilidad de PMN y la pureza son&gt; 99% según la evaluación por exclusión de azul de tripano y la visualización de la morfología nuclear después de la tinción, respectivamente. </li></ol> 5. Transuroepithelial neutrófilos Migración Comodecir Realice los siguientes pasos en una campana de cultivo de tejidos utilizando una técnica aséptica. Utilice sólo soportes permeables teniendo confluentes 5637 capas de células, tal como se determina en el paso 2.7. Las placas de 24 pocillos que contienen medio RPMI-se pueden preparar por adelantado y se mantuvieron a 37 ° C con 5% de CO 2 hasta su uso. <ol><li> Alícuota 1 ml calentar RPMI-por pocillo en una placa de 24 pocillos, preparar 3 pocillos por soporte permeable. </li><li> Aspirar el medio desde los depósitos superior e inferior de los soportes permeables. </li><li> Usando una pinza estéril, transferir los soportes permeables a la placa de 24 pocillos preparada en el paso 5.1. Lavar los soportes permeables tres veces mediante la transferencia de los soportes de un pozo a otro. </li><li> Si un inóculo bacteriano se va a utilizar, invertir los soportes permeables en un 25 mm de placa de cultivo de tejido profundo estéril. Si un quimioatrayente (por ejemplo, N-formil-Met-Leu-Phe (fMLF) o IL-8) es para ser utilizado, continúe en el paso 5.6. </li><li> Para experimentos con bact vivo o muertoestímulos erial, inocular el lado apical de cada soporte permeable con 60 l de RPMI-(infección simulada) o con el inóculo bacteriano (6 x 10 6 UFC) preparado en la etapa 3.5. Coloque la tapa en la placa y se incuba a 37 ° C con 5% de CO 2 durante 1 hora. </li><li> Añadir 0,6 ml de RPMI-por pocillo a una placa de 24 pocillos de bajo apego; preparar 1 bien por soporte permeable. Preparar 3 pocillos que contenían 0,5 ml de RPMI-enumerar entrada PMN. <ol><li> Si un quimioatrayente se está utilizando en lugar de bacterias, añadir el quimioatrayente a 0,6 ml de RPMI-en la placa de 24 pocillos preparada en la etapa 5.6. </li></ol></li><li> El derecho de los soportes permeables en la placa de 24 pocillos de bajo apego. </li><li> Añadir 0,1 ml de PMN (10 6 PMN), preparado en la etapa 4.9, con el depósito superior (lado basolateral) de cada soporte permeable. Añadir 100 l de PMN directamente a los pocillos que contienen 500 l de RPMI-. Incubar a 37 ° C con 5% de CO 2 durante 1 hora. </li><li> Usando una pinza estéril, raspar suavemente elmembrana del soporte permeable contra el borde del pozo para eliminar los PMN adicional de la parte apical y luego disponer del soporte. </li><li> Recoger PMN raspando suavemente la parte inferior de cada pocillo con la punta de pipeta 1.000 l, y la transferencia de las suspensiones de PMN a Microfuge tubos. </li><li> Enumerar PMN usando un hemocitómetro. </li><li> Para calcular el número total de PMN en el depósito inferior, se multiplica el número de PMN en 1 mm 2 (100 nl) de 6000. Los datos pueden ser reportados como un porcentaje de los PMN de entrada, o como números de PMN normalizaron a 10 6 PMN de entrada. </li></ol>

<ol>
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W., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+urinary+tract+infection.">A murine model of urinary tract infection.</a> Nat. Protoc. 4, 1230-1243 (2009).</li></ol>

Los autores no tienen nada que revelar.

Usando una línea de células epiteliales cultivadas de la vejiga y recién aisladas PMN humanos, establecimos un modelo in vitro de la migración de neutrófilos transuroepithelial. Este modelo ha sido instrumental en el inicio de diseccionar la complejidad de la respuesta inmune innata durante la infección del tracto urinario (ITU), una infección bacteriana muy común por lo general causada por la UPEC 9. Durante la infección de la vejiga, o cistitis, el reclutamiento de los PMN al lumen de la v...

El movimiento de las células en todo el cuerpo, a menudo a través de largas distancias, se requiere para el crecimiento y el desarrollo, la curación de heridas, y la respuesta inmune. La migración celular es complejo y requiere la coordinación de muchos procesos diferentes, incluyendo cascadas de señalización y la reordenación de los componentes del citoesqueleto. Las células pueden moverse al azar (quimiocinesis), así como hacia gradientes químicos definidos (quimiotaxis). Muchas técnicas se han desarrollado para estudiar la migración celular in vitro. La técnica más antigua y más común, la cámara de Boyden, consiste en un sistema de dos cámaras verticales cuando una sustancia quimiotáctica se coloca en la cámara inferior y células de interés se colocan en la cámara superior 1. El movimiento de las células a través del filtro permeable, con poros de tamaño definido, que separa las dos cámaras se controla. Otras técnicas se han desarrollado para investigar la migración de células, incluyendo la cámara de Zigmond 2 y la cámara de Dunn3. Estos enfoques colectivos han aportado una información valiosa sobre el movimiento de muchos tipos de células diferentes. Además de interrogar a los principios básicos de la quimiocinesis y la quimiotaxis, ensayos de dos cámaras han facilitado la investigación de la migración celular a través de componentes de la matriz extracelular y ambas capas de células endoteliales y epiteliales. Una ventaja de los sistemas de dos cámaras sobre otras técnicas es que la membrana porosa se puede recubrir con proteínas tales como colágeno o fibrinógeno, y la migración de células a través de una barrera de matriz extracelular se puede evaluar. Además, las líneas de células cultivadas se pueden cultivar y diferenciar en los soportes permeables. Para investigar el movimiento de las células a través de una barrera endotelial, células endoteliales cultivadas se siembran y cultivan en el depósito superior de los soportes permeables. Células móviles, tales como las células inmunes, se añaden al depósito superior y la migración en el depósito inferior a través de la linease observa barrera endotelial en la dirección fisiológica-apical a basolateral. Este modelo ha sido muy valioso para la comprensión de la extravasación de las células inmunes de la sangre. En contraste con la migración transendotelial, el movimiento de las células a través de una barrera epitelial se produce normalmente en la dirección basolateral a apical. Con el fin de modelar estos eventos in vitro, los investigadores siembran y crecen las células epiteliales cultivadas en la parte inferior de los soportes permeables. Células móviles se añaden al depósito superior y la migración a través de la barrera epitelial, que representa la dirección basolateral a apical, se supervisa. Estos modelos de la migración transepitelial han contribuido significativamente a nuestra comprensión de las respuestas inflamatorias en las superficies de las mucosas, especialmente los de pulmón y el intestino 4,5. En contraste, el tráfico de células inmune a través del epitelio de las vías urinarias ha recibido mucha menos atención. Para avanzar en nuestra understanding de la respuesta inmune innata en el tracto urinario durante la infección con uropatógena de Escherichia coli (UPEC), hemos desarrollado un ensayo in vitro, el ensayo de migración de neutrófilos transuroepithelial, que permite la investigación de leucocitos polimorfonucleares (PMN; neutrófilos) movimiento a través de una barrera epitelial de la vejiga 6 -8. Al igual que con otros modelos de dos cámaras de la migración transepitelial, células epiteliales de la vejiga humanas cultivadas se cultivan en la parte inferior de un soporte permeable y forman capas epiteliales confluentes. Los neutrófilos humanos, aislados a partir de sangre venosa, se aplican a la parte basolateral de la capa epitelial, y la migración a través del epitelio en la dirección basolateral a apical fisiológicamente relevante se cuantifica en respuesta a la infección con diferentes cepas de E. coli o la presencia de moléculas quimioatrayentes. Mucha investigación se ha centrado en el movimiento de los neutrófilos, tanto chemokinesis y quimiotaxis, en ausencia de otros tipos de células. El ensayo de la migración de neutrófilos transuroepithelial es ventajoso, ya que toma en cuenta las interacciones complejas entre las células epiteliales de la vejiga y de células inmunes durante la infección. Este modelo tratables in vitro tiene el potencial de permitir la investigación detallada de la respuesta inmune en superficies uroepiteliales.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Transwell Inserts (i.e.&#160;permeable supports)</td>
			<td>Corning</td>
			<td>3472</td>
			<td>6.5 mm, 3 &#956;m pore, polyester membrane insert</td>
		</tr>
		<tr>
			<td>BD Vacutainer Blood Collection Tubes</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>366480</td>
			<td>16 mm x 100 mm x 10 ml green cap tubes containing sodium heparin</td>
		</tr>
		<tr>
			<td>BD Safety-Lok Blood Collection and Infusion Set</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>367281</td>
			<td>21 G x 0.75 in needle x 12 in tubing</td>
		</tr>
		<tr>
			<td>BD Vacutainer one-use, nonstackable holder</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>364815</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>D4876</td>
			<td>From Leuconostoc mesenteroides</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS density centrifugation solution</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Plates</td>
			<td>Corning</td>
			<td>3473</td>
			<td>24-well, clear flat bottom</td>
		</tr>
		<tr>
			<td>N-Formyl-Met-Leu-Phe (fMLF)</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td>Reconstituted in DMSO to 10 mM</td>
		</tr>
		<tr>
			<td>Recombinant Human CXCL8/IL-8</td>
			<td>R&#38;D Systems</td>
			<td>208-IL</td>
			<td>Reconstituted in PBS to 100 &#956;g/ml</td>
		</tr>
	</tbody>
</table>

quantitative assessment of human neutrophil migration across a cultured bladder epithelium

El reclutamiento de células inmunes desde la periferia hacia el sitio de la inflamación es un paso esencial en la respuesta inmune innata en cualquier superficie de la mucosa. Durante la infección de la vejiga urinaria, los leucocitos polimorfonucleares (PMN; neutrófilos) migran desde el torrente sanguíneo y atraviesan el epitelio de la vejiga. La incapacidad de resolver la infección en ausencia de una respuesta neutrofílica demuestra la importancia de los PMN en la defensa de la vejiga. Para facilitar la colonización del epitelio de la vejiga, uropatógena de Escherichia coli (UPEC), el agente causante de la mayoría de las infecciones del tracto urinario (ITU), amortiguar la respuesta inflamatoria aguda utilizando una variedad de mecanismos parcialmente definidas. Para investigar más a fondo la interacción entre el huésped y el patógeno bacteriano, hemos desarrollado un modelo in vitro de este aspecto de la respuesta inmune innata a la UPEC. En el ensayo de migración de neutrófilos transuroepithelial, una variación en la cámara de Boyden, epith vejiga cultivadasElial células se cultivan hasta la confluencia en la parte inferior de un soporte permeable. PMN se aíslan a partir de sangre venosa humana y se aplica a la cara basolateral de las capas de células epiteliales de la vejiga. Migración de PMN que representa la dirección basolateral a apical fisiológicamente relevantes en respuesta a la infección bacteriana o moléculas quimioatrayentes se enumera usando un hemocitómetro. Este modelo se puede utilizar para investigar las interacciones entre la UPEC y células eucariotas, así como para interrogar a los requisitos moleculares para el recorrido de los epitelios de la vejiga por los PMN. El modelo de la migración de neutrófilos transuroepithelial será mejorar nuestra comprensión de la respuesta inflamatoria inicial a la UPEC en la vejiga.

El ensayo de migración de neutrófilos transuroepithelial permite la evaluación cuantitativa de la migración de PMN humano a través de capas de células epiteliales de la vejiga cultivadas en respuesta a diversos estímulos (Figura 1B). Mientras que el protocolo es sencillo, hay un número de variables que pueden influir en la migración de PMN y por consiguiente afectar a la reproducibilidad de este ensayo. Se deberían tomar medidas, mientras que la preparación de los soportes permeables y el PMN...

Watch this Scientific Journal Video about Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium at JoVE.com

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

Hemos desarrollado un modelo in vitro que imita un componente importante de la respuesta inflamatoria aguda durante la infección de la vejiga con Escherichia coli uropatógena. El ensayo de migración de neutrófilos transuroepithelial permite la evaluación cuantitativa de la migración de neutrófilos humanos a través de los epitelios de la vejiga, se cultivaron en soportes permeables, en respuesta a la infección bacteriana o sustancias quimioatrayentes.

Evaluación cuantitativa de neutrófilo humano migración a través de una vejiga cultivada Epitelio

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

quantitative-assessment-human-neutrophil-migration-across-cultured

Research

JoVE Journal

Immunology and Infection

14.9K vistas.  Washington University School of Medicine.  Hemos desarrollado un modelo in vitro que imita un componente importante de la respuesta inflamatoria aguda durante la infección de la vejiga con Escherichia coli uropatógena. El ensayo de migración de neutrófilos transuroepithelial permite la evaluación cuantitativa de la migración de neutrófilos humanos a través de los epitelios de la vejiga, se cultivaron en soportes permeables, en respuesta a la infección bacteriana o sustancias quimioatray...

Video: Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansión, purificación y evaluación funcional de la sangre periférica humana células NK

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Investigación

Inmunología e Infección

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Seguimiento de Rastreo de neutrófilos intraluminal, la migración transendotelial y la quimiotaxis de los tejidos por microscopía de vídeo intravital

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Una evaluación cuantitativa de la migración celular mediante el ensayo de la canción Motilidad Phagokinetic

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Cuantitativo<em&gt; In vitro</em&gt; Ensayo para medir la adhesión de neutrófilos al Activado Primaria células endoteliales microvasculares humanas en condiciones estáticas

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Ensayo de cocultivo para evaluar la migración transepitelial de neutrófilos inducida por patógenos

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Ensayo de adhesión de neutrófilos Cámara de flujo humano

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

El desarrollo y la identificación de una subpoblación de Novela derivada de neutrófilos humanos fagocitos gigante<em&gt; In Vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In Vitro Formación de la trampa extracelular neutrófilo canino: Análisis dinámico y cuantitativo por microscopía de fluorescencia

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

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 ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

Evaluación Funcional De La Permeabilidad Intestinal Y La Migración Transepitelial Neutrófilo En Ratones Usando Un Modelo De Bucle Intestinal Estandarizado

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...