La inducción de DTH adoptivos Por favor, consulte el artículo Jove: <a href="/index/Details.stp?ID=325">Inducción y Seguimiento de la Adopción de hipersensibilidad retardada en ratas</a> Christine Beeton, George K. Chandy Departamento de Fisiología y Biofísica de la Universidad de California, Irvine J. visualizados experimentos, Número 8 Aquí, hemos modificado este protocolo mediante el antígeno (ovoalbúmina) conjugado en Texas-Red en una proporción 1:1 con antígeno sin marcar para permitir la visualización de la fagocitosis de las células presentadoras de antígeno. Cosecha del tejido del oído <ol><li> La eutanasia a la rata en el tiempo deseado de puntos por los procedimientos descritos en el protocolo de animales aprobada. Aquí, nosotros usamos una sobredosis de anestésico, el isoflurano. Esto se hace en un recipiente cerrado ubicado dentro de una campana bien ventilada. Asegúrese de que el animal está muerto, ya sea mediante la apertura de la decapitación de la cavidad torácica y cortar el corazón. </li><li> Con la tijera grande quitar la oreja con un solo corte, tan cerca del cuerpo del animal como sea posible (Figura 1). <img src="/files/ftp_upload/old/907_2008_8_13_0/Figure_1.jpg" alt="Figure_1.jpg" /> Figura 1. La eliminación de la oreja de rata </li><li> Colocar el oído en un tubo cónico de 15 ml contiene aproximadamente 10 ml de helado de 1x PBS o RPMI 1640. Mantenga las muestras de tejido en el hielo hasta que esté listo para preparar el tejido para obtener imágenes. </li><li> La imagen del tejido, tan pronto como sea posible, nos encontramos, sin embargo, que 1 a 2 horas sobre el hielo no tiene ningún efecto discernible sobre la motilidad celular en el tejido en comparación con los tejidos cosechados y la imagen de inmediato. </li></ol> Preparación para el tejido de imágenes I. La eliminación de la capa epidérmica y dérmica * Nota: La capa dérmica profunda debe mantenerse intacto. Si se hace correctamente los grandes vasos sanguíneos se mantendrá intacto y el cartílago de la oreja no serán expuestos. Esta es una técnica difícil, especialmente cuando no hay inflamación (las condiciones de control) y es útil usar un microscopio de disección. <ol><li> Lugar tanto en el tejido y los medios de comunicación desde el tubo cónico en una placa de cultivo de 10 cm de tejido. </li><li> Con las manos enguantadas mantener el oído rata suavemente en la punta de la oreja (distal) con la cara dorsal de la oreja hacia arriba (Figura 2). <img src="/files/ftp_upload/old/907_2008_8_13_1/Figure_2.jpg" alt="Figure_2.jpg" /> Figura 2. Recorte de piel de la oreja </li><li> Con el pulgar en la parte superior de la oreja, constante, mientras que el tejido con la punta de cierre Dumont # 5 tijeras para separar la dermis de la dermis profunda. Alternativamente, si el borde de la oreja tiene un saliente de la piel / dermis profunda, esto puede ser captado con unas pinzas y se saca suavemente para separar el tejido por debajo de la (Figura 3). <img src="/files/ftp_upload/old/907_2008_8_13_2/Figure_3.jpg" alt="Figure_3.jpg" /> Figura 3. La eliminación de la dermis superior </li><li> Una vez que una pequeña área de piel ha sido separada del tejido subyacente, repita los pasos de separación, alternativamente, utilizando tijeras para cortar entre las capas de la dermis y pinzas para quitar con cuidado y retire la piel desalojados. </li><li> Exponer un 5 x 5 mm o mayor área de tejido del oído que es por lo menos 3 mm desde el sitio inicial de la inyección de antígeno (Figura 4). <img src="/files/ftp_upload/old/907_2008_8_13_3/Figure_4.jpg" alt="Figure_4.jpg" /> Figura 4. Tejido expuesto de imágenes </li></ol> II. Asegurar el tejido en el escenario de imágenes <ol><li> Cortar una hoja de cubierta de plástico de un tamaño ligeramente mayor que el tejido preparado. </li><li> Extender una fina capa de 3M ™ Vetbond tejido de seguridad de cola a través de la diapositiva. </li><li> Sujete la esquina del tejido preparado (en la que no es probable que la imagen), retirar de los medios de comunicación, y quite la parte inferior del tejido en el papel lente o Kym borrar para eliminar el exceso de material. </li><li> Tocar el borde del tejido a la diapositiva cola cubierta (el cura Vetbond inmediatamente en el tejido húmedo). Estira suavemente el tejido plano, con el extremo opuesto está el último en tocar la diapositiva (Figura 5) <img src="/files/ftp_upload/old/907_2008_8_13_4/Figure_5.jpg" alt="Figure_5.jpg" /> Figura 5. Monte tejido para cubrir deslizamiento </li><li> Curar la ™ Vetbond invirtiendo la muestra de tejido / diapositivas y la introduce en un depósito de los medios de comunicación de tal manera que el tejido y se deslizan hacia abajo y son más o menos en un ángulo de 45 grados. Sosteniendo el tejido al revés en un ángulo ayuda a prevenir el exceso de pegamento de secado en la superficie de los tejidos expuestos. Pegamento en la superficie del tejido se bloqueará el láser (Figura 6). <img src="/files/ftp_upload/old/907_2008_8_13_5/Figure_6.jpg" alt="Figure_6.jpg" /> Figura 6. Cure vetbond tejido glue por inmersión en los medios de comunicación Nota: los pasos 2-5 puede tomar un poco de práctica para dominar. Trate de practicar con trozos de tejido desechado o más antes de intentar por primera vez. </li><li> Aplique una pequeña cantidad de grasa de silicona en la parte inferior de la diapositiva. </li><li> Colocar la placa en la cámara de perfusión. Asegúrese de que los medios de comunicación de perfusión está fluyendo, 37 ° C y oxigenada antes de añadir tejido. Presione suavemente los bordes de la diapositiva haciendo un sello con la grasa de silicona entre la parte inferior de la cámara de perfusión y la hoja de la cubierta de plástico. </li></ol> III. De dos fotones de imágenes <ol><li> El uso de campo brillante luz transmitida, se centran en la parte superior del tejido. Apague todas las luces. </li><li> Encienda el láser, PMT según lo requiera el sistema de microscopía multi-fotón. </li><li> Generación de segundo armónico (SHG) en las proteínas fibrosas altamente ordenadas (colágeno y miosina) produce una señal en la mitad de la longitud de onda del láser de excitación. Nosotros usamos la excitación a 900 nm, que lleva a los grupos de autoayuda a 450 nm, que pueden ser visualizados a través del &quot;azul&quot;, el canal del microscopio. SHG generación es un no-lineales (de dos fotones) de procesos, y por lo tanto ofrece una óptica de &quot;corte&quot; efecto similar al de la excitación de dos fotones de fluorescencia. La máxima eficiencia de los grupos de autoayuda se produce cuando las fibras son perpendiculares a la láser, mientras que las fibrillas que se ejecutan en paralelo no se visualiza 1. </li><li> Localizar un área de imagen buena con un montón de células, establece el área de adquisición de tal manera que la mayoría de las células están en el centro y comenzar a imágenes. Nota: En las reacciones de DTH, las células T y se infiltra en otras células se encuentran a menudo se concentran en conjunto, mientras que otras áreas están desprovistas de la infiltración de células 2. Por lo tanto, puede tomar algún tiempo buscando el tejido para encontrar la mejor zona de la imagen. </li><li> Compruebe que la viabilidad celular mediante la evaluación de la velocidad y la polaridad celular durante la exploración. Usa el poder más de láser que proporciona una calidad de imagen aceptable para asegurar un mínimo de foto-daño. </li><li> Swap en una preparación de tejido nuevo necesario. </li></ol>

<ol>
	<li>Koo, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+the+voltage-gated+potassium+channel+Kv1.3+inhibits+immune+responses+in+vivo.">Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.</a> J Immunol. 158, 5120-5128 (1997).</li><li>Gaga, M., Frew, A. J., Varney, V. A., Kay, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eosinophil+activation+and+T+lymphocyte+infiltration+in+allergen-induced+late+phase+skin+reactions+and+classical+delayed-type+hypersensitivity.">Eosinophil activation and T lymphocyte infiltration in allergen-induced late phase skin reactions and classical delayed-type hypersensitivity.</a> J Immunol. 147, 816-822 (1991).</li><li>Flugel, A., Odoardi, F., Nosov, M., Kawakami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoaggressive+effector+T+cells+in+the+course+of+experimental+autoimmune+encephalomyelitis+visualized+in+the+light+of+two-photon+microscopy.">Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy.</a> J Neuroimmunol. 191, 86-97 (2007).</li><li>Kawakami, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+effector+cell+trafficking+and+autoantigen+recognition+within+the+unfolding+autoimmune+encephalomyelitis+lesion.">Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion.</a> J Exp Med. 201, 1805-1814 (2005).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Matheu, M. P. B., A, C. G. a. r. c. i. a., Chi, V., Rangaraju, S., Safrina, O., Monaghan, K., Uemura, M. I., Li, D., Pal, S., Monuki, E., Flugel, A., Pennington, M. W., Parker, I., Chandy, G. K., Calahan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Imaging+of+Effector/Memory+T+Cells+During+DTH+and+Suppression+by+Kv1.3+Channel+Block.">In Situ Imaging of Effector/Memory T Cells During DTH and Suppression by Kv1.3 Channel Block.</a> Immunity. , (2008).</li></ol>

imaging effector memory t cells in the ear after induction of adoptive dth

Retardada de hipersensibilidad (DTH) es una reacción inmune en el cual los principales actores son CCR7 - efectoras / memoria los linfocitos T. En este caso, se demuestra un método para inducir y registrar el progreso de una reacción de DTH en el oído de la rata. Esto es seguido por una demostración de la elaboración de tejidos de ratas oído para la imagen de dos fotones de la CCR7 - respuesta efectoras / memoria de células T. Un DTH adoptivos es inducida por la inyección intraperitoneal de las buenas prácticas agrarias con etiqueta específica OVA-CCR7 - línea efectoras / memoria de células T (Beeton, J. C experimentos visualizados, número 8). Células se dejaron equilibrar en la rata durante 48 horas antes de la exposición mediante la inyección de una oreja con una solución salina (control del oído) y el otro con una mezcla de 1:1 de conjugado OVA y OVA de Texas-Red (Ova-TR) para permitir la visualización residente de células presentadoras de antígeno. Se describe un método de preparación de tejidos útiles para obtener imágenes de la motilidad de las células en la capa de la dermis profunda durante una respuesta inmune, en combinación con la visualización de las fibras de colágeno por la generación del segundo armónico. Tejido del oído se corta en cuadrados de 5 mm x 5 (ligeramente más grande es mejor) y montado sobre cubreobjetos de plástico con Vetbond ™, que luego se fija con grasa de silicona en una cámara de imágenes y superfused por burbujas de oxígeno medio de cultivo de tejidos a 37 ° C .

Watch this Scientific Journal Video about Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH at JoVE.com

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Aquí se demuestra un método para inducir y registrar el progreso de un retraso de tipo de hipersensibilidad (DTH) la reacción en el oído de la rata. Esto es seguido por una demostración de la elaboración de tejidos de ratas oído para la imagen de dos fotones de la respuesta del efector / memoria de células T.

Imágenes de las células efectoras T de memoria en el oído después de la inducción de DTH adoptivos

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

imaging-effector-memory-t-cells-ear-after-induction-adoptive

Research

JoVE Journal

Biology

10.2K vistas.  University of California, Irvine (UCI). Aquí se demuestra un método para inducir y registrar el progreso de un retraso de tipo de hipersensibilidad (DTH) la reacción en el oído de la rata. Esto es seguido por una demostración de la elaboración de tejidos de ratas oído para la imagen de dos fotones de la respuesta del efector / memoria de células T.

Video: Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

Inducción y seguimiento de activos de hipersensibilidad de tipo retardado (DTH) en ratas

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Investigación

Biología

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Inducción y Seguimiento de Adopción de hipersensibilidad retardada en ratas

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria y electroporación plásmido del órgano de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Ensayo clonigénicas: las células adherentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&amp;#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Imágenes de los receptores de estrógenos-α en ratas arteriolas Pial usando un microscopio de inmunofluorescencia Digital

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagen Ca Local<sup&gt; 2 +</sup&gt; Las señales en las células de mamífero en cultivo

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Una interfaz gráfica de usuario para el seguimiento asistido por software de la concentración de proteínas en protones dinámicos celulares

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Disección eficiente y cultivo de células epiteliales de pigmento retiniano de ratón primario

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...