El trabajo presentado en este artículo fue apoyado por un Nacional de Salud y Consejo de Investigación Médica (NHMRC) de Australia Grant Program (CRP) y un proyecto NHMRC Grant (CRP y BJCQ). También BJCQ es un NHMRC Peter Doherty Postdoctoral Fellow.

1) El reactivo de configuración 1.1) CFSE solución madre El tinte CFSE se compra como diacetato de carboxifluoresceína éster de succinimidilo (CFDA, SE) (MW 557,47 g / mol), por lo general en viales de 25 mg. Disolver la dosis de 25 mg en 8,96 ml de DMSO para una solución de reserva final de 5 mM (50-100 almacenar alícuotas a -20 ° C durante varios meses). CFDA, SE va a reaccionar con una solución acuosa por lo que es fundamental que dicha exposición evitarse durante el almacenamiento. 1.2) Los linfocitos Aislar linfocitos de bazo y los ganglios linfáticos o de los ratones y resuspender en 1 ml de medio de cultivo (por ejemplo RPMI) con 5% inactivado por calor (HI) de suero fetal bovino (FCS), con una concentración celular de 0,5 x 10 06 al 10 x 10 7 / ml. 2) CFSE etiquetado 2.1) de rutina método: <ol class="lalpha"><li> Bien volver a suspender las células en el volumen de 1 ml de medio y colocar cuidadosamente en el fondo de un fresco (no mojado) tubo cónico de 10 ml. </li><li> Coloque el tubo en posición horizontal (utilizando un tubo que no impedirá que el contacto con el medio 1 ml de células de suspensión de movimiento y mezcla antes de tiempo con el CFDA soluciones, SE). </li><li> Añadir con cuidado 110 l de PBS a la porción no-en contacto con el plástico en la parte superior del tubo de modo que no pueda hacer contacto con la solución de células. </li><li> Resuspender 1,1 l de la población de 5 mm de CFDA, SE en el PBS 110 mL. </li><li> Rápidamente tapar el tubo e invierta y agite bien para conseguir uniformes rápida mezcla de las soluciones. Tenga en cuenta que el tinte CFSE se encuentra en una concentración final de 5 mM, sin embargo, la concentración óptima puede tener que ser determinado para cada lote preparado, y luego cada 6 meses para asegurar que sea efectivo. </li><li> Se incuban las células durante 5 minutos a temperatura ambiente. Tenga en cuenta que la conversión de la CFDA, SE CFSE (véase la discusión) se convierte en el tinte fluorescente y por tanto, susceptibles de alterarse por la exposición excesiva a la luz. Por lo tanto, es recomendable proteger el tubo de la luz a partir de ahora, por ejemplo, cubriéndola con papel de aluminio. </li><li> Lavar las células mediante la dilución en 10 volúmenes de 20 ° C PBS con 5% FCS HI, sedimentación por centrifugación a 300 xg durante 5 min a 20 º C y desechar el sobrenadante. Repetir el lavado dos veces más. </li></ol> 2.2) Método alternativo, que también es apropiado para el número de células más alta (10 - 30 x 10 7): <ol class="lalpha"><li> Preparar un 2 x (10 M) CFDA, la solución de la SE por la adición de 2 l de la población de 5 mM a 1 ml de PBS y añadir rápidamente esta a 1 ml de células resuspendidas a fondo, y rápidamente se tapa el tubo e invierta y agite bien para conseguir uniforme de la mezcla rápida. </li><li> Se incuban las células durante 5 minutos a temperatura ambiente y se lava como en los pasos 2.1 (f) y 2.1 (g). </li></ol> 2.3) Las células se pueden aplicar a un protocolo de interés para la inducción de la división celular. Células marcadas se pueden utilizar en los estudios in vitro y en ensayos in vivo. 2.4) A la finalización del ensayo de proliferación, las células se recogen y se analizan por citometría de flujo (ver más abajo para los ejemplos representativos). 3) Resultados de Representante Para evaluar la proliferación de linfocitos por dilución CFSE, es útil conocer la fluorescencia de las células indivisas (es decir, el máximo de fluorescencia) y no marcado células (fluorescencia es decir, como mínimo). Por lo tanto, el diseño experimental debe tomar en cuenta estos controles. A continuación se muestran ejemplos de ensayos in vitro y en ensayos in vivo utilizando CFSE para medir CD8 + división de las células T por citometría de flujo. 3.1) En la estimulación in vitro La figura 1 muestra los perfiles de los CFSE CFSE marcado con ovoalbúmina (OVA) específicos de las células T CD8 + receptor transgénicos OT-I las células T después de la cultura 3 días con células dendríticas pulsadas con diversas cantidades de OVA. Células T CD8 + purificados de los ganglios linfáticos y el bazo de los ratones OT-I fueron marcadas con CFSE y 1 x 10 5 células en cultivo con 3,3 x 10 4 DC. DC, donde pulsada con OVA durante 1 hora a 37 º C y lavado dos veces antes de la cultura. Después de 3 días, las células donde se cosechan y se marcaron con anti-CD8-APC anticuerpos y Hoechst 33258 y luego analizadas por citometría de flujo (50.000 eventos recopilados). Live (Hoechst 33258 -) las células CD8 + se muestran. Como los controles, las células CD8 + T cultivadas solas, muestra la intensidad de la CFSE no dividen las células, mientras que las células no llevan la etiqueta muestra la autofluorescencia de las células y los límites de las divisiones celulares detectables. Tenga en cuenta que cuatro picos CFSE se puede ver en cada uno de los paneles en este ejemplo, lo que indica que las células han sido sometidos a un máximo de 3 divisiones. 3.2) En la estimulación in vivo La figura 2 muestra los perfiles de la CFSE CFSE marcado con OVA-específico de los receptores de células T CD8 + transgénicos OT-I las células T después de 3 días en vivo in la presencia de OVA 20 mg. Células T CD8 + purificados de los ganglios linfáticos y el bazo de CD45.1 congenic OT-I ratones fueron marcadas con CFSE y 5 x 10 6 células inyectadas iv en la vena lateral de la cola de C56BL/6J (CD45.2 +) ratones. Después de 2 horas los ratones fueron inyectados iv con 20 mg de OVA. Después de 3 días, el bazo de los ratones han sido recogidos, hecho en una única suspensión de células, etiquetados con anti-B220-PerCP, anti-CD8-APC-Cy7 y anti-CD45.1-PE-Cy7 anticuerpos y Hoechst 33258 y se analizan por citometría de flujo (1.000.000 sucesos recogidos). Live (Hoechst 33258 -) B220-las células se muestran en el panel de la izquierda y cerrado CD8 + células CD45.1 + muestra en el panel derecho. Como controles, sin estimular CFSE marcado con células T CD8 +, muestra la intensidad de la CFSE no dividen las células, mientras que los no etiquetados células muestra la autofluorescencia de las células y los límites de las divisiones celulares detectables. Tenga en cuenta que el uso de la diferencia CD45 alotípicos es vital en la resolución del transferido células T CD8 + de acogida, ya que son un subconjunto menor de edad del total de células T CD8 + (panel izquierdo). Tenga en cuenta que la mayoría de las células caen dentro de los 7 picos CFSE en este ejemplo (panel derecho), lo que indica que las células han sido sometidos a un máximo de 6 divisiones. <img src="/files/ftp_upload/2259/2259fig1.jpg" alt="Figura 1" /> Figura 1. Capacidad de los CFSE marcado con ovoalbúmina (OVA) específicos de las células T del receptor de transgénicos CD8 + OT-I las células T para responder in vitro para DC pulsado con diferentes concentraciones de OVA, los datos muestran que después de 3 días de cultivo. Tenga en cuenta que no divide la población de linfocitos T CD8 + las células en ausencia de antígeno (la luz histograma gris) y auto-fluorescencia de la población no etiquetados (histograma de color gris oscuro). La figura muestra células T CD8 + que han dividido a 1-3 veces sobre la base de los picos de dilución CFSE, constituido por células T que divide a las concentraciones de antígeno superior. <img src="/files/ftp_upload/2259/2259fig2.jpg" alt="Figura 2" /> Figura 2. Capacidad de los CFSE marcado con ovoalbúmina (OVA) específicos de las células T del receptor de transgénicos CD8 + OT-I las células T, cuando adoptively transferido en ratones C57BL / 6 para responder a OVA, datos que se muestran tres días después de la transferencia adoptiva Panel izquierdo:. CD8 +, CD45. 1 + OT-1 de células T en ratones receptores. Panel de la derecha: el perfil de CFSE proliferación. Tenga en cuenta que no divide la población de células T CD8 + en los ratones receptores no reciben el antígeno (la luz histograma gris) y auto-fluorescencia de los no etiquetados linfocitos (histograma de color gris oscuro). La figura muestra las células CD8 + T que han dividido a 1.6 veces sobre la base de los picos de dilución CFSE. <img src="/files/ftp_upload/2259/2259fig3.jpg" alt="Figura 3" /> Figura 3. Una representación de los diversos eventos moleculares que ocurren durante el etiquetado de las células de diacetato de carboxifluoresceína éster de succinimidilo (CFDA, SE). Para más detalles sobre el proceso de ver el texto de discusión. Nota: &quot;CFSE&quot; El acrónimo genérico, no CFDA, SE, se utiliza para describir el reactivo de marcaje y el procedimiento de etiquetado en general. Figura basada en la parroquia 4.

<ol>
	<li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171, 131-137 (1994).</li><li>Parish, C. R., Glidden, M. H., Quah, B. J., Warren, H. S., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+intracellular+fluorescent+dye+CFSE+to+monitor+lymphocyte+migration+and+proliferation.">Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.</a> Current protocols in immunology. , (2009).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nature Protocols. 2, 2049-2056 (2007).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77, 499-508 (1999).</li></ol>

Con el fin de apreciar cómo funciona y por qué CFSE etiquetado uniforme, rápido se requiere para su uso en el análisis de la proliferación, es útil para entender la base molecular de la CFSE etiquetado de la célula. Esto puede ser dividido en dos fases, 1) entrada de celda y 2) el etiquetado de proteínas (Figura 3). 1) entrada a la célula: la entrada del colorante en la célula es mediado por la forma diacetilados del colorante, diacetato de carboxifluoresceína succinimidil éster (CFDA, SE). Los acetatos que el colorante permeable membrana que permite su flujo muy rápido a través de la membrana plasmática. Esterasas, presentes en la célula, unirá los acetatos de CFDA, SE y por lo tanto dar lugar a la forma de la CFSE tinte que es mucho menos permeable la membrana, lo que se concentra el medio de contraste dentro de la célula. 2) El etiquetado de proteínas: Mientras tanto CFDA, SE y CFSE han amino-reactiva cadenas laterales succinimidilo, sólo la CFSE es fluorescente. La alta concentración intracelular de la CFSE facilita el etiquetado rápido y de alto nivel intracelular de proteínas. CFSE etiquetado de las células debe realizarse rápidamente a fin de obtener una población homogénea de células etiquetadas, lo cual es fundamental para la resolución de las células que han sufrido varias divisiones como se muestra en los resultados representativos (Figura 1 y 2).

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<td>CFDA,SE</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>DMSO</td>
<td>&nbsp;</td>
<td>Cambridge Isotope Laboratories Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Lymphocytes</td>
<td>&nbsp;</td>
<td>eg mouse or human</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>RPMI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>SAFC Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10-15ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon, Becton Dickinson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Scientific Industries</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
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the use of carboxyfluorescein diacetate succinimidyl ester (cfse) to monitor lymphocyte proliferation

Carboxifluoresceína succinimidil ester (CFSE) es un medio eficaz y popular para controlar la división de linfocitos 1-3. CFSE etiquetas covalentemente moléculas de larga vida intracelular con el colorante fluorescente, carboxifluoresceína. Por lo tanto, cuando una célula se divide CFSE marcado, todos sus descendientes están dotados de la mitad del número de carboxifluoresceína etiquetados con las moléculas y por lo tanto cada división celular se puede evaluar midiendo la correspondiente disminución de la fluorescencia celular mediante citometría de flujo. La capacidad de la CFSE para etiquetar las poblaciones de linfocitos, con una alta intensidad de fluorescencia de la varianza excepcionalmente bajo, unido a su baja toxicidad celular, lo convierten en un medio de contraste ideal para medir la división celular. Ya que es un colorante de fluoresceína basado también es compatible con una amplia gama de fluorocromos otros por lo que es aplicable a la citometría de flujo multi-color. En este artículo se describen los procedimientos normalmente utilizados para el etiquetado de linfocitos de ratón con el propósito de monitorear hasta ocho divisiones celulares. Estas células marcadas se pueden utilizar tanto para la in vitro e in vivo.

Watch this Scientific Journal Video about The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation at JoVE.com

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE etiquetas covalentemente moléculas de larga vida intracelular con el colorante fluorescente, carboxifluoresceína. Por lo tanto, cuando una célula se divide CFSE marcado, todos sus descendientes tienen la mitad de la cantidad de fluorescencia, que de ese modo se puede utilizar para evaluar la división celular. En este artículo se describen los procedimientos normalmente utilizados para el etiquetado de linfocitos de ratón con CFSE.

El uso de diacetato de carboxifluoresceína Ester succinimidil (CFSE) para controlar la proliferación de linfocitos

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

the-use-carboxyfluorescein-diacetate-succinimidyl-ester-cfse-to

Research

JoVE Journal

Immunology and Infection

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

91.3K vistas.  John Curtin School of Medical Research, Australian National University. CFSE etiquetas covalentemente moléculas de larga vida intracelular con el colorante fluorescente, carboxifluoresceína. Por lo tanto, cuando una célula se divide CFSE marcado, todos sus descendientes tienen la mitad de la cantidad de fluorescencia, que de ese modo se puede utilizar para evaluar la división celular. En este artículo se describen los procedimientos normalmente utilizados para el etiquetado de linfocitos de ratón con CFSE.

Video: The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Punción en el dedo de sangre Metodología de muestreo para la determinación de la inducida por el ejercicio apoptosis de los linfocitos

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

Investigación

Inmunología e Infección

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Un ensayo visual para monitorear T6SS mediada por la competencia bacteriana

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

El uso de<em&gt; En vivo</em&gt; Imágenes para controlar la progresión de la infección por citomegalovirus Experimental del ratón en neonatos

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

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 ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

La inmunofluorescencia para supervisar la captación celular de lactoferrina humana y su Asociado actividad antiviral contra el virus de hepatitis C

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

El uso de la citometría de flujo para evaluar el estado de la cromatina en las células T

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Ensayo de adhesión en condiciones de estrés de cizalla para el Estudio de linfocitos T-molécula de adhesión Interacciones

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Detección de<em&gt; Trypanosoma brucei</em&gt; Variant glicoproteína de superficie de conmutación por Magnetic Activated Cell Sorting y Citometría de Flujo

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Desarrollo de un sistema reportero virus de la hepatitis B para supervisar las primeras etapas del ciclo de replicación

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...