The authors thank Ian Clarke and P. Scott Hefty for the pGFP-SW2 and pASK-GFP-mKate2-L2 plasmids, respectively. We thank Paul Miller for technical assistance and Richard Stephens for other resources. This work was funded by NIH grant AI095603 (KH).

1. Generación de host fluorescente Líneas Celulares <ol><li> Día 1. Las células 293T Plate (u otra línea de envasado retroviral) en placas de 6 pocillos a 2 x 10 6 células / Bueno, para ser ~ 75% confluentes al día siguiente. Si se desea, la placa de pocillos duplicados para cada uno de retrovirus a utilizar. </li><li> Día 2. células Aspirar y añadir 2 ml de medio de cultivo fresco (DMEM + 10% FBS + 2 mM L-glutamina). Transfectar células con 5-8 g cada uno de vector retroviral (que contiene GFP) y envasado vector (por ejemplo., PVSVg) usando Lipofectamine 2000 o reactivo similar y siguiendo las instrucciones del fabricante. </li><li> Se incuban las células con el ADN durante 4-6 horas en un incubador de CO2 C 37 °, y posteriormente sustituir medio con medio de crecimiento 1 ml. </li><li> Se incuban las células durante 48 horas en 37 ° C incubadora de CO 2. </li><li> Día de las células 3. Placa de destino (por ejemplo., HeLa) en placas de 6 pocillos a una densidad aproximada de 10 5 células / pocillo, para ser ~ 50% de confluencia deldía siguiente. Verificar la transfección positiva en células 293T mediante el control de la fluorescencia de GFP en un microscopio invertido. </li><li> Día 4. Recoger sobrenadantes fuera de las células 293T con una pipeta y filtrar el sobrenadante a través de un filtro de jeringa de acetato de 0,45 micras de celulosa que contienen retrovirus. </li><li> Retire medio a partir de células HeLa y añadir medio de crecimiento de 0,5 ml contiene 16 mg / ml de polibreno. Añadir ~ 0,5 ml de retrovirus filtrada a las células gota a gota e incubar las células en una incubadora de CO 2 C 37 ° durante 24 horas. Almacenar cualquier retrovirus restantes (es decir, desde el duplicar pocillo) a 4 ° C. </li><li> Día 5. Si se hicieron pocillos duplicados para generar retrovirus adicional, repita el paso 1.7 para infectar serie con las partículas retrovirales restantes. </li><li> Día 6. Passage células HeLa transfectadas 1: 1 en una placa de 10 cm que contiene la selección de antibióticos (por ejemplo, 500 mg / ml de neomicina). </li><li> Espere el tiempo suficiente para que las células transducidas a crecer de nuevo en presencia de la selección antibiotic, después de lo cual las células se pueden propagar mediante técnicas estándar con una concentración más baja de antibiótico de selección (por ejemplo., 200 mg / ml de neomicina). Nota: Las células también se pueden seleccionar clonalmente para el brillo ideales en este momento, si es necesario. </li></ol> 2. Generación de GFP-expresión de Chlamydia trachomatis <ol><li> Preparar 2x CaCl 2 tampón (20 mM Tris pH 7,4, 100 mM CaCl 2) y tampón 4SP (0,4 M sacarosa, 16 mM Na 2 HPO 4). </li><li> Día 1. Descongele congelada C. trachomatis disponible el hielo e inmediatamente diluir 10 l bacterias en 50 l 2x CaCl tampón 2. Añadir 3 ADN plásmido mg (por ejemplo., PASK-GFP-mKate2-L2), mezclar bien e incubar durante 30 minutos a 25 ° C. </li><li> Durante este paso, preparar células huésped mediante tripsinización un matraz T-75 de células McCoy en un tubo de centrífuga. Centrifugar la suspensión celular a 50 xg durante 5 min y descartar el sobrenadante. Resuspend celular pellet en volumen apropiado de tampón 1x CaCl 2 para dar una concentración final de 4 x 10 7 células / ml. </li><li> Después de la incubación de 30 min (de la etapa 2.2), añadir 100 l preparados células McCoy para tubo de mezcla de transformación (volumen total 200 l) y se incuba un 20 min adicionales a 25 ° C. Añadir 100 l de esta mezcla de células transformadas a un solo pocillo de una placa de 6 pocillos y superposición con medio 2 ml crecimiento. Incubar a 37 ° C en una incubadora de CO 2 durante 2 días. </li><li> Día 3. células Lyse McCoy infectadas con C. trachomatis mediante la sustitución de medio con 1 ml de dH 2 O y raspado de células con una punta de pipeta de 1.000 l doblada. Añadir 1 ml de tampón 4SP y pasar la suspensión a través de una aguja de 27,5 G ~ 5 veces para la lisis celular eficiente y liberación de C. trachomatis. </li><li> Transferencia de 2 ml del lisado celular que contiene C. trachomatis a un matraz T-75 que contiene una monocapa confluente de recién chapado McCcélulas oy; añadir 8 ml de DMEM (sin FBS) y se incuba durante 2 horas a 25 ° C para permitir C. trachomatis a que se adhieran a las células. </li><li> Células Aspirar y añadir medio de crecimiento fresco suplementado con 10 U / ml de penicilina G y 1 mg / ml de cicloheximida. Incubar matraz durante 48 h en una incubadora de 37 ° C CO 2. </li><li> Día 4. Verifique por microscopía de luz que las células están infectadas y inclusiones contienen Chlamydia aberrante. Identificar inclusiones como vacuolas brillantes en un microscopio óptico estándar, y los organismos de Chlamydia aberrantes como objetos de anillo dentro de inclusiones que son mayores de 2 m de diámetro. </li><li> Día 5. cultura Lyse reemplazando medio con 2 ml dH2O y raspar las células en una suspensión. Añadir 2 ml de tampón 4SP y pasar la suspensión a través de una aguja de 27,5 G ~ 5 veces. </li><li> Utilice 2 ml de lisado de células para infectar una monocapa fresca de células McCoy en un solo pocillo en una placa de 6 pocillos. Se incuban las células con lisado durante 2 horas a 25 ° C, y añadir aspirado de crecimiento frescomedio que contiene penicilina G y cicloheximida. </li><li> Se incuban las células en una incubadora a 37 ° C CO 2 durante 3-5 días, dependiendo de la salud general de las células. </li><li> Repita los pasos 2.9-2.10 una o más veces, según sea necesario, culturas pases en pozos frescas en una placa de 6 pocillos hasta inclusiones buscan normales (desprovistos de cuerpos aberrantes) han surgido. En este momento utilizar un microscopio de fluorescencia invertida para comprobar que C. trachomatis expresar mKate2 (rojo). </li><li> Ampliar los cultivos infectados para la generación de las poblaciones de clamidias alto título, por ejemplo, mediante la recolección de transformantes Chlamydia de células McCoy o HeLa infectadas cultivadas en matraces T-150. </li></ol> 3. La infección de células con Chlamydia <ol><li> Placa células GFP-HeLa en recipiente de cultivo deseada, por ejemplo, platos de vidrio de fondo o la cámara de diapositivas de imágenes de alta resolución, o placas de 24 pocillos para la determinación IFU y la detección de múltiples pocillos. </li><li> Descongele frotubo de zen que contiene C. trachomatis, C. muridarum, o C. pneumoniae en hielo y diluido en HBSS a la multiplicidad deseada de infección (MOI), por ejemplo MOI = 1. </li><li> Realizar infecciones ya sea estáticas o centrifugación asistido basado en la cepa particular Chlamydia que se utilizará. <ol><li> Para las infecciones con C. trachomatis LGV serovar L2 o C. muridarum, lavar las células GFP-HeLa con HBSS y se incuba con bacterias diluidas durante 2 horas a 25 ° C. Aspirar y lavar las células con HBSS, e incubar las células en medio de crecimiento en 37 ° C incubadora de CO 2. </li><li> Para las infecciones con C. trachomatis serovar D o C. pneumoniae, lavar las células GFP-HeLa con HBSS y añadir bacterias diluidas a las células. Coloque la placa de múltiples pocillos o plato de fondo de cristal (garantizado por la cinta en una tapa de placa de múltiples pocillos) en un soporte de la placa de múltiples pocillos de un accesorio de rotor basculante en una centrífuga de mesa. </li><li> Células centrifugar a 900 xg a 25 ° C durante 1 hora. </li&gt; <li> Retire recipientes de cultivo de centrífuga y el lugar en una incubadora a 37 ° C CO 2 durante 1 hora. </li><li> Aspirar las células en un gabinete de bioseguridad, se lavan las células dos veces con HBSS, y añadir medio de cultivo fresco. Coloque las células en un incubador de CO2 C 37 °. </li></ol></li></ol> 4. Visualización de células vivas de Chlamydia Inclusiones <ol><li> Retire Chlamydia infectadas células GFP-HeLa de CO 2 incubadora y reemplazar medio con RPMI sin rojo fenol + 5% de SFB. </li><li> Células de montaje en un microscopio de fluorescencia invertido (Nikon Eclipse Ti-E o similar) usando un objetivo 20X o superior petróleo. </li><li> El uso de software de imagen (Volocity 6 o similar), se centran en identificar y clamidia infecta células GFP-HeLa: células verdes que contienen grandes agujeros negros (inclusiones de Chlamydia). </li><li> Marque las ubicaciones xyz de las regiones que contienen las células infectadas por clamidia de interés utilizando software de imagen (Volocity 6 o similar). Realizar esto manualmente &quot;Añadir puntos &#39;, mientras que en el modo de vista XY escenario. De esta manera, las coordenadas xyz se guardan para cada ubicación marcada. </li><li> En el cuadro de diálogo Configuración de Adquisición, configurar el software para adquirir verde (GFP, HeLa citosol) y rojo (mKate2, C. trachomatis) canales, ya una tasa de nuevos cuadros por canal cada 5 min. </li><li> Adquirir la secuencia de imágenes mediante la adquisición de protocolo entró anterior haciendo clic en el botón &quot;Capture / Record&quot;. </li></ol> 5. La cuantificación de los parámetros de crecimiento de inclusión en células vivas <ol><li> En tiempos deseados después de la infección (por ejemplo., 24, 48 hpi) eliminar clamidia infecta células GFP-HeLa de la incubadora y montar en un microscopio de fluorescencia invertida. Nota: Un objetivo 20X o 40X aire con un alto NA es más adecuado para las placas de 24 pocillos, y se recomienda un objetivo de aceite de 40X para la cámara de diapositivas. Crecer células a cada sustrato para este culoay. </li><li> Concéntrese en planos medios de las células, donde las inclusiones están en su diámetro máximo y producir bordes nítidos. </li><li> Adquirir imágenes de muchos campos en cada pocillo (por lo menos 10 por pocillo); pozos normalmente representan réplicas o variables experimentales. </li><li> Enumerar el número de inclusiones de forma manual, por el ojo (inclusiones son compartimentos negros en células GFP-HeLa) o utilizar algoritmos de software de formación de imágenes para identificar y contar automáticamente las inclusiones. <ol><li> Encuentra células GFP-HeLa por la intensidad de fluorescencia (&#39;Encuentra Objetos&#39; comando) y ajustar la intensidad umbral de fluorescencia basada en intensidades relativas de las células. </li><li> Filtrar objetos seleccionados utilizando directrices de tamaño de mantener sólo aquellos mayores de 5 micras 2. Se trata de &#39;población 1&#39;. </li><li> Aplicar &#39;Rellenar agujeros en objetos&#39; comando utilizando &#39;población 1&#39; como entrada. Este paso genera &#39;población 2&#39;. </li><li> Reste &#39;población 1&#39; de &#39;población 2 &#39;para dar marcó positivamente inclusiones de Chlamydia, designados como &quot;población 3&#39;. </li><li> Aplicar filtro de tamaño de &quot;población 3 &#39;(comando&#39; Filtro Población &#39;), por ejemplo, los objetos de mantenimiento mayor que 25 m 2 para inclusiones en células infectadas durante 24 horas o más. Para tiempos de infección temprana, como antes de la 18 horas, filtro de tamaño ajustado para mantener los objetos de más de 4 m 2, ya que estas inclusiones son mucho más pequeños. Resultados de filtrado Tamaño de una población de objetos designados como &quot;inclusiones&quot;. </li><li> Calcular datos cuantitativos a partir de imágenes, por ejemplo, número de la inclusión, el tamaño de la inclusión, y la circunferencia de la inclusión, utilizando software de imágenes. </li></ol></li></ol>

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+expression+in+Chlamydia+trachomatis+using+the+tet+system.">Conditional gene expression in Chlamydia trachomatis using the tet system.</a> PLoS One. 8 (10), 10-1371 (2013).</li><li>Chin, E., Kirker, K., Zuck, M., James, G., Hybiske, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+recruitment+to+the+Chlamydia+inclusion+is+spatiotemporally+regulated+by+a+mechanism+that+requires+host+and+bacterial+factors.">Actin recruitment to the Chlamydia inclusion is spatiotemporally regulated by a mechanism that requires host and bacterial factors.</a> PLoS One. 7 (10), e46949 (2012).</li><li>Agaisse, H., Derr&#233;, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+trachomatis+cloning+vector+and+the+generation+of+C.+trachomatis+strains+expressing+fluorescent+proteins+under+the+control+of+a+C.+trachomatis+promoter.">A C. trachomatis cloning vector and the generation of C. trachomatis strains expressing fluorescent proteins under the control of a C. trachomatis promoter.</a> PLoS One. 8 (2), e57090 (2013).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Aquí se describe la estrategia experimental para la generación de células huésped fluorescentes para visualización en tiempo real y análisis de inclusiones de Chlamydia. Este enfoque visualización vacuola confiere la poderosa capacidad de iluminar, rastrear y cuantitativamente medir las propiedades dinámicas de inclusiones clamidias través de las poblaciones de células o en células individuales en el tiempo. Inclusiones de Chlamydia en las células de proteínas etiquetados fluorescentes est...

Las enfermedades infecciosas causadas por especies de la bacteria Chlamydia intracelular provocan una carga importante en la salud global, incluyendo enfermedades de transmisión sexual, enfermedad inflamatoria pélvica, la ceguera, neumonía y posiblemente aterosclerosis 1.4. La capacidad de Chlamydia para interactuar con la célula huésped, desde dentro de una vacuola (denominada la inclusión), es un determinante crítico para su éxito de la infección de las células y el anfitrión. La inclusión es una novela compartimento patógeno que permite el crecimiento por clamidias y se modifica dinámicamente durante todo el ciclo de desarrollo de 2-3 días de Chlamydia 5. La naturaleza intracelular obligado de clamidias presenta numerosos retos para la comunidad de investigación, en particular para estudiar directamente de la biología única de la inclusión. Un obstáculo importante ha sido la incapacidad de visualizar de manera eficiente ya sea Chlamydia intracelular o su inclusión por el enfoque fluorescenteES en células vivas. Un descubrimiento reciente ha revelado por fin los medios para generar GFP expresando C. trachomatis 6; Sin embargo, este hallazgo aún no ha dado lugar a un etiquetado específico de la inclusión. Algunas técnicas se han descrito para el etiquetado de las bacterias y las inclusiones 7,8, pero adolecen de deficiencias tales como la no especificidad, la transitoriedad y la susceptibilidad a photobleaching. Un descubrimiento clave de nuestro grupo estableció una nueva estrategia para la iluminación de la inclusión mediante las buenas prácticas agrarias que expresan las células anfitrionas 9. Esta estrategia explota racionalmente la impermeabilidad intrínseca de la inclusión de membrana a las moléculas de mayor que 520 Da 10. Cuando las células están diseñados para expresar de forma estable una proteína fluorescente citosólica particular (por ejemplo, las buenas prácticas agrarias o mCherry), inclusiones de Chlamydia son visibles con notable claridad por su exclusión completa de fluorescencia. Esta estrategia de inversión de imágenes permite la visualización inmediata de inclusiones para todos Chlespecies amydia y que se pueden adaptar fácilmente para la mayoría de las células huésped de interés. Como demostración de su utilidad, este método fue utilizado anteriormente para revelar y definir las vías de salida celulares para Chlamydia spp 9. Aquí, una vez más demostrar cómo se realiza este procedimiento, y puede ser explotado para obtener datos cuantitativos clave acerca de la dinámica de crecimiento de inclusión. Además, se puede sustituir con eficacia de costosos métodos de enumeración basadas en anticuerpos y se puede utilizar en conjunto con otras etiquetas fluorescentes, tales como mKate2-expresión de Chlamydia 11. Esta poderosa combinación de herramientas permite la exploración de las propiedades físicas de la membrana inclusión clamidia dentro de las células anfitrionas de estar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-Mem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filters</td>
			<td>Fisher</td>
			<td>09-719D</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>10131</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o phenol red</td>
			<td>Cellgro</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Sigma</td>
			<td>13752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber slides, Lab-Tek II</td>
			<td>Nunc</td>
			<td>154526, 154534</td>
			<td></td>
		</tr>
	</tbody>
</table>

using fluorescent proteins to visualize and quantitate chlamydia vacuole growth dynamics in living cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Las células de mamífero que expresan proteínas fluorescentes citosólicas (por ejemplo, GFP) pueden ser diseñados para permitir la iluminación de inclusiones de Chlamydia en cultivos de células en vivo, infectados. Tras la infección con Chlamydia, las inclusiones son fácilmente visibles como puntos negros en las células huésped (Figura 1). La claridad de las inclusiones de fluorescencia de carecer puede ser explotado para la identificación automática de las inclusi...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells at JoVE.com

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

El uso de proteínas fluorescentes para visualizar y cuantificar<em&gt; Chlamydia</em&gt; Vacuola dinámica de crecimiento en las células vivas

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

using-fluorescent-proteins-to-visualize-quantitate-chlamydia-vacuole

Research

JoVE Journal

Immunology and Infection

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

7.5K vistas.  University of Washington. A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Video: Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Investigación

Inmunología e Infección

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Siete pasos para células estrelladas

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A raíz de la celda de destino en<em&gt; E. coli</em&gt; Después de la infección por fagos Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

Growth La medición y de la Expresión Génica Dinámica de Tumor-Targeted<em&gt; S. Typhimurium</em&gt; Las bacterias

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

Enfoques Forward genéticos en<em&gt; Chlamydia trachomatis</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Vigilar los cambios en la polaridad de la membrana, membrana Integridad, e intracelular de iones concentraciones en<em&gt; Streptococcus pneumoniae</em&gt; Uso de tintes fluorescentes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

El uso de proteínas fluorescentes de Seguimiento glicosoma Dinámica en el tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Fagosoma Cierre de ensayo&quot; para visualizar Fagosoma Formación en tres dimensiones utilizando la reflexión interna total fluorescente Microscopía (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Precisión de corte pulmón de ratón Rebanadas para visualizar las células en vivo pulmonar dendríticas

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.