Esta investigación fue apoyada por la División de Investigación Intramural del Instituto Nacional de Alergias y Enfermedades Infecciosas, Institutos Nacionales de Salud. Estamos muy agradecidos a Juan Huppa y Mark Davis para que nos proporciona el scFv de H57 se utilizan en estos estudios. RV gustaría dar las gracias Keir Neumann útil para el debate durante el desarrollo de esta técnica.

<ol>
	<li>Call, M. E., Wucherpfennig, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T+cell+receptor:+critical+role+of+the+membrane+environment+in+receptor+assembly+and+function.">The T cell receptor: critical role of the membrane environment in receptor assembly and function.</a> Annu. Rev. Immunol. 23, 101-125 (2005).</li><li>Smith-Garvin, J. E., Koretzky, G. A., Jordan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S.T+cell+activation.">S.T cell activation.</a> Annu. Rev. Immunol. 27, 591-619 (2009).</li><li>Bezbradica, J. S., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+cytokine+and+heterologous+receptor+signaling+pathways.">Integration of cytokine and heterologous receptor signaling pathways.</a> Nat. Immunol. 10, 333-339 (2009).</li><li>Fraser, I. D., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigating+the+network:+signaling+cross-talk+in+hematopoietic+cells.">Navigating the network: signaling cross-talk in hematopoietic cells.</a> Nat. Immunol. 10, 327-331 (2009).</li><li>Dustin, M. L., Starr, T., Varma, R., Thomas, V. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor-proximal+signals+are+sustained+in+peripheral+microclusters+and+terminated+in+the+central+supramolecular+activation+cluster.">T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.</a> Immunity. 25, 117-127 (2006).</li><li>Yokosuka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+generated+T+cell+receptor+microclusters+initiate+and+sustain+T+cell+activation+by+recruitment+of+Zap70+and+SLP-76.">Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.</a> Nat. Immunol. 6, 1253-1262 (2005).</li><li>Huppa, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR-peptide-MHC+interactions+in+situ+show+accelerated+kinetics+and+increased+affinity.">TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.</a> Nature. 463, 963-967 (2010).</li><li>Tolar, P., Pierce, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conformation-induced+oligomerization+model+for+B+cell+receptor+microclustering+and+signaling.">A conformation-induced oligomerization model for B cell receptor microclustering and signaling.</a> Curr. Top. Microbiol. Immunol. 340, 155-169 (2010).</li><li>Treanor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+skeleton+controls+diffusion+dynamics+and+signaling+through+the+B+cell+receptor.">The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor.</a> Immunity. 32, 187-199 (2010).</li><li>Treanor, B., Batista, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insight+into+lymphocyte+activation+through+quantitative+imaging+and+theoretical+modelling.">Mechanistic insight into lymphocyte activation through quantitative imaging and theoretical modelling.</a> Curr. Opin. Immunol. 19, 476-483 (2007).</li><li>Sylvain, N. R., Nguyen, K., Bunnell, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vav1-mediated+scaffolding+interactions+stabilize+SLP-76+microclusters+and+contribute+to+antigen-dependent+T+cell+responses.">Vav1-mediated scaffolding interactions stabilize SLP-76 microclusters and contribute to antigen-dependent T cell responses.</a> Sci. Signal. 4, ra14-ra14 (2011).</li><li>Purbhoo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+subsynaptic+vesicles+and+surface+microclusters+at+the+immunological+synapse.">Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse.</a> Sci. Signal. 3, ra36-ra36 (2010).</li><li>Lasserre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ezrin+tunes+T-cell+activation+by+controlling+Dlg1+and+microtubule+positioning+at+the+immunological+synapse.">Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse.</a> EMBO. J. 29, 2301-2314 (2010).</li><li>Balagopalan, L., Sherman, E., Barr, V. A., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+assaying+lymphocyte+activation+in+action.">Imaging techniques for assaying lymphocyte activation in action.</a> Nat. Rev. Immunol. 11, 21-33 (2011).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+7:+Total+internal+reflection+fluorescence+microscopy.">Chapter 7: Total internal reflection fluorescence microscopy.</a> Methods Cell. Biol. 89, 169-221 (2008).</li><li>Williamson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-existing+clusters+of+the+adaptor+Lat+do+not+participate+in+early+T+cell+signaling+events.">Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events.</a> Nat. Immunol. 12, 655-662 (2011).</li></ol>

Todos los animales utilizados en estos experimentos se mantuvieron en un libre de agentes patógenos medio ambiente, y los experimentos fueron aprobados por los Institutos Nacionales de Salud Animal y el empleo.

Se describe aquí un sistema de señalización en el estudio de antígenos específicos de células T primarios de ratón utilizando TIRFM vidrio y apoyadas bicapas lipídicas artificiales como vehículos blindados. La técnica se basa en las buenas prácticas agrarias con éxito la expresión de proteínas etiquetadas en estas células. Transfección de células T es siempre una tarea difícil. Típicamente, la entrega de electroporación o genes utilizando retrovirus o lentivirus se utilizan. Una técnica no es superior sobre el otro, ambos tienen sus limitaciones y ventajas. Encontramos que la electroporación tiene las siguientes ventajas: 1) No se requiere que las células se dividen activamente como se requiere para los retrovirus, pero no lentivirus, y 2) El nivel de expresión puede ser controlada mediante la variación del tiempo, las células se incuban antes de imágenes. El mayor inconveniente es que nos resulta muy difícil expresar las proteínas que son de gran tamaño en las células primarias. Hemos presentado un método que nos da la viabilidad celular muy bueno después de la electroporación. Como resultado, es serían aplicables-le a su utilización para lograr mediada por siRNA silenciamiento de los genes. También describe aquí un personalizado dos canales simultáneos microscopio TIRF adquisición que se cromáticamente corregida y no requiere alineación separado para diferentes longitudes de onda. Estas capacidades, sin embargo, están disponibles comercialmente. Nuestro sistema se basa en los principios de la microscopía TIRF publicado por expertos en la materia y es más barato que las alternativas comerciales. Una posible crítica de nuestro diseño es que estamos usando el mismo ángulo de incidencia para las longitudes de onda de excitación diferentes. Esto daría lugar a una profundidad de penetración diferente TIRF para longitudes de onda de excitación diferentes. Creemos que estos efectos son pequeños debido a que la onda evanescente es un campo de forma exponencial en descomposición y la profundidad del campo TIRF es una función lineal de longitud de onda. Fluoróforos cercanas a la superficie del vidrio experimentará ninguna diferencia en la intensidad de láser entre las dos longitudes de onda. Para fluoróforos cerca de la PEnetration profundidad, la diferencia de intensidad será 1,3 veces para las dos longitudes de onda 488 y 640 nm y plegado 1,15 para las dos longitudes de onda 488 nm y 561. El mismo sistema puede ser usado en conjunción con técnicas de resolución súper que hacen uso de iluminación TIRF. También se ha descrito un sistema de dos cámaras, que es hecha a la medida. Al igual que el sistema TIRF, aparatos similares también están disponibles comercialmente. Nuestro sistema ofrece flexibilidad para cambiar los filtros dicroicos y, lo que permite su uso con diferentes combinaciones de longitudes de onda. El inconveniente de nuestro sistema es el grado de una rotación necesaria para alinear las dos imágenes. Este problema se puede resolver mediante el uso de soportes de la cámara que se piezo impulsado y ofrecen grados de rotación de la alineación. También hemos aplicado con éxito un sistema de cámara dos en este microscopio, en la que las cámaras son diferentes y tienen diferentes tamaños de pixel. Un sistema como este podría ser útil si se necesita la sensibilidad en un canal y un gran dyrango dinámico en otro, los cuales rara vez están disponibles en la misma cámara. Se utilizó la Fotometría HQ-2 cámara en binning 2x2 que nos da un tamaño de píxeles efectivos de 12,9 m con la Fotometría Quant-EM cámara que tiene un tamaño de píxel de 16 micras. Un 180 mm de longitud focal de lente del tubo se utiliza para la Quant-EM y un 145 mm de longitud focal del objetivo tubo fue utilizado para la cámara HQ-2. El HQ-2 se controla mediante el software Metamorph y el Quant-EM se controla a través de software de micro-manager en un equipo distinto en el modo de disparo externo. El disparo externo se proporciona a la cámara Quant-EM con una salida digital de la medición a bordo del CAD de computación, que se llevó a cabo como un disparador adicional en la configuración de configure iluminación de Metamorph. La relación de las distancias focales de las lentes del tubo coincide con la proporción de los tamaños de pixel eficaces. Una alineación representativo se muestra en la Figura 5.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Amaxa nucleofector Kit de ratón las células T </td><td> Lonza Inc. </td><td> VPA-1006 </td><td></td></tr><tr><td> PureLink HiPure plásmido Kit Midiprep </td><td> Tecnologías de la Vida </td><td> K2100-05 </td><td> Para preparaciones de ADN libres de endotoxinas </td></tr><tr><td> Amaxa nucleofector dispositivo </td><td> Lonza Inc. </td><td> AAD-1001 </td><td></td></tr><tr><td> Murina recombinante de IL-2 </td><td> Peprotech Inc. </td><td> 212-12 </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent microesferas 0.20μm </td><td> Polysciences, Inc. </td><td> 24050-5 </td><td></td></tr><tr><td> Lab-Tek II de 8 cámaras y cubre objetos </td><td> Thermo Scientific, Nunc </td><td> 155409 </td><td></td></tr></tbody></table>

a tirf microscopy technique for real-time, simultaneous imaging of the tcr and its associated signaling proteins

La señalización se inicia a través del receptor de la célula T (TCR) cuando se acopla con fragmentos de péptidos antigénicos unidos por complejo mayor de histocompatibilidad (pMHC) las proteínas expresadas en la superficie de las células presentadoras de antígeno (APC). El complejo TCR se compone del heterodímero ligando TCRαβ unión que asocia de forma no covalente con CD3 dímeros (los εδ y εγ heterodímeros y el homodímero ζζ) 1. Tras la participación del receptor, las cadenas ζ de CD3 se fosforilada por la quinasa de la familia Src, Lck. Esto conduce a la contratación de la familia Syk quinasa, Zap70, que luego se fosforila y activa por Lck. Después de eso, Zap70 fosforila el adaptador de proteínas LAT y SLP76, iniciando la formación del complejo de señalización proximal que contiene un gran número de diferentes moléculas de señalización 2. La formación de este complejo, finalmente, los resultados en calcio y TRA Ras-dependientenscription activación de los factores y la iniciación consecuente de una compleja serie de programas de expresión de genes que dan lugar a la diferenciación de células T 2. Señales (TCR y el estado resultante de la diferenciación) son modulados por otros muchos factores, incluyendo la potencia del antígeno y la diafonía con co-stimulatory/co-inhibitory, quimiocinas y receptores de citoquinas 3-4. El estudio de la organización espacial y temporal del complejo de señalización proximal en condiciones de estimulación diferentes, por lo tanto, clave para entender la vía de señalización del TCR, así como su regulación por otras vías de señalización. Uno de los sistemas modelo muy útil para estudiar la señalización iniciada por el TCR en la membrana plasmática de las células T es de cristal apoyados por bicapas de lípidos, como se describe anteriormente 5-6. Ellos pueden ser utilizados para presentar complejos antigénicos pMHC, adherencia, y moléculas co-estimuladoras a las células T-sirviendo como APCs artificiales. Por imágenes de las células T interactuar con elbicapa lipídica utilizando total de microscopía de fluorescencia interna reflexión (TIRFM), se puede restringir la excitación a 100 nm dentro del espacio existente entre el cristal y la superficie de la célula 7-8. Esto nos permite la imagen sobre todo los eventos de señalización que ocurren en la membrana plasmática. Como estamos interesados ​​en la imagen de la contratación de las proteínas de señalización del complejo TCR, se describe una de dos cámaras TIRF sistema de imagen en la que el TCR, marcado con fluorescente de Fab (fragmento de unión del antígeno) fragmentos del anticuerpo H57 (purificada a partir de hibridoma H57-597 , ATCC, número ATCC: HB-218) que es específico para TCRβ, y proteínas de señalización, marcado con GFP, pueden tomarse imágenes simultáneamente y en tiempo real. Esta estrategia es necesaria debido a la naturaleza altamente dinámica tanto de las células T y de los eventos de señalización que se están produciendo en el TCR. Esta técnica de imagen ha permitido a los investigadores a los ligandos de imágenes individuales 9-11, así como el reclutamiento de moléculas de señalización de los receptores activados yes un excelente sistema para estudiar la bioquímica in situ 12-16.

Watch this Scientific Journal Video about A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins at JoVE.com

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

La compartimentación de las proteínas, ya sea dentro de la membrana plasmática o en lugares intracelulares es un mecanismo de regulación que pueden influir mucho en los resultados de señalización, por lo que, para comprender la señalización es importante estudiar el comportamiento espacial y temporal de las proteínas implicadas. Se describe aquí un sistema de microscopía TIRF base para estudiar la transducción de señal en las células T, pero es ampliamente aplicable.

Una técnica de microscopía TIRF en tiempo real, imágenes simultáneas de la TCR y sus proteínas asociadas de señalización

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

a-tirf-microscopy-technique-for-real-time-simultaneous-imaging-tcr

Research

JoVE Journal

Immunology and Infection

23.7K vistas.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. La compartimentación de las proteínas, ya sea dentro de la membrana plasmática o en lugares intracelulares es un mecanismo de regulación que pueden influir mucho en los resultados de señalización, por lo que, para comprender la señalización es importante estudiar el comportamiento espacial y temporal de las proteínas implicadas. Se describe aquí un sistema de microscopía TIRF base para estudiar la transducción de señal en las células T, pero es ampliamente a...

Video: A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Imágenes en tiempo real de los leucotrienos B 4 La migración mediada por células y BLT1 interacciones con β-arrestina

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Investigación

Inmunología e Infección

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

En tiempo real de imágenes en vivo de las células T de señalización Formación de complejos

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Imágenes en tiempo real de la célula-célula cruces endoteliales Durante neutrófilos Transmigración Bajo Flujo Fisiológica

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Imágenes en tiempo real de las células mieloides en Dinámica<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumores de Spinning disco microscopía confocal

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

La cuantificación simultánea de T-Cell Círculos Receptor Excision (TRECs) y Círculos K-Eliminación recombinación Excision (KRECs) por PCR en tiempo real

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Análisis de<em&gt; Yersinia enterocolitica</em&gt; Efector translocación en las células huésped Usando beta-lactamasa Efector fusiones

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging neutrófilos y monocitos en mesentérica venas por Microscopia intravital en ratones anestesiados en Tiempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Purificación de ADN Viral para la identificación de las proteínas virales y celulares asociadas

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Imagen en tiempo real y cuantificación del desarrollo de biopelículas fúngicas mediante un sistema recirculación de flujo bifásico

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

Procesamiento de imagen protocolo para el análisis de la difusión y del tamaño del clúster de receptores de membrana por microscopía de fluorescencia

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...