We would like to thank the Children&#8217;s Hospital at Montefiore, Department of Pediatrics, for providing support for these studies. We would like to thank the National Alopecia Areata Foundation for providing financial support for conducting studies with C3H/HeJ engrafted mice.

Declaración de Ética: Todos los estudios con animales deben ser aprobados por el IACUC de la institución (por los datos que siguen, los protocolos fueron aprobados por el Montefiore IACUC, protocolo # 11-6-240 y # 13-7-100). Animales se proporcionan anestesia luz como se indica con la única finalidad de mantenerlos quietos durante la fotografía, no hay procedimientos dolorosos requeridos para este protocolo. 1. Adquisición de fotografías <ol><li> Ajuste el enfoque y campo de visión para reproductor de imágenes de gel utilizando papel con el texto impreso. Verificar la uniformidad de la fuente de luz en toda la región fotografiada. Para ayudar a asegurar la uniformidad, utilice un generador de imágenes de gel con un sistema incorporado en la fuente de luz para la fotografía reflexiva. No utilice una fuente de luz transiluminación, ya que esto crearía una silueta del animal que no es adecuado para su posterior análisis en escala de grises. NOTA: Esto colocará el ratón ligeramente fuera de foco, que proporciona un promedio óptica a través de la región de interés (ROI) y la voluntadayudar a reducir los errores cuánticos para regiones muy pequeñas (es decir, &lt;10 píxeles) de interés. Regiones más grandes de interés no se verán afectados por esta menor centrándose ajuste, ni serán afectados por las diferencias en el tamaño del animal. </li><li> Anestesiar a los animales, utilizando ketamina (100 mg / ml) / xilazina (20 mg / ml) (2: 1), ya que esto proporciona un efecto de la anestesia rápida y recuperación rápida y es óptimo para fotografiar múltiples animales. NOTA: La anestesia se confirmó cuando el animal es todavía suficiente para permitir la fotografía. Como los animales se recuperan de esta luz anestesia dentro de 10-15 minutos, el veterinario no ha recomendado el uso de ungüentos para los ojos. </li><li> Coloque los animales anestesiados en gel de imágenes en alineación vertical (tan cerca como sea posible a paralelo). <ol><li> Para las fotografías dorsales, colocar los animales en decúbito prono, con los miembros extendidos. </li><li> Para las fotografías ventrales, colocar los animales en decúbito supino, con cuidado de que los animales no se rotan lateralmente. </li&gt; </ol></li><li> Coloque estándar en escala de grises en la región fotografiada. </li><li> Cierre la puerta de acceso. Importante: La luz ambiental puede introducir variaciones en la exposición. </li><li> Establecer F-stop a una exposición que sitúa a la región de interés dentro del rango lineal de adquisición (lectura F-parada). NOTA: La mayoría de los sistemas se mostrarán donde la imagen está saturado. </li><li> Tome la fotografía. </li><li> Cambio F-parar a otro ajuste de exposición que sitúa a la región de interés dentro del rango lineal aumentando o disminuyendo la ventanilla F por 1, de manera que tanto la norma y la región de interés se mantienen en un rango lineal de adquisición (referencia F- deténgase). </li><li> Tome la fotografía. </li><li> Una vez que la fotografía se ha completado, colocar los animales en una mesa de calentamiento y monitorear hasta que puedan mantener decúbito esternal. Vuelta de los animales a sus jaulas. Devuelva el grupo de animales para el vivero, una vez que todos los animales están completamente recuperados. </li></ol> 2. Cuantificación de absorción de la luz &lt;/ P&gt; <ol><li> Marcar las regiones de interés en las imágenes de los animales utilizar el software proporcionado por imager gel. <ol><li> Para vista dorsal animal completo, utilice una imagen rectangular u oval que se extiende desde los miembros superiores a las extremidades inferiores, que se extiende lateralmente tanto como sea posible de tal manera que ninguna parte de la caja se extiende más allá de la parte posterior del animal, como se muestra en la Figura 1A. NOTA: También se podría marcar el área de interés utilizando una herramienta de dibujo a mano alzada. </li><li> Para el conjunto de vista ventral animal, utilizar 2 rectángulos: uno que cubre la región pélvica y uno que cubre la zona del pecho, como se muestra en la Figura 1B. </li><li> Para una región más pequeña de interés, es decir, la ubicación de la administración del fármaco, marca, según corresponda. </li></ol></li><li> Marcos región (s) de interés en el estándar de absorción de escala de grises. </li><li> Absorción registro de cada una de las regiones marcadas de interés. </li></ol> 3. Análisis Niveles de absorción obtained para las regiones de interés puede necesitar ser normalizados a la norma de fondo para la comparación entre las fotografías. La relación de la exposición a la absorción es log-log (es decir, la relación es lineal entre log (exposición) y log (absorción)). Usando esta relación, la absorción de la región de interés puede ser normalizado a un valor de fondo estándar, que permitirá absorciones que deben compararse directamente entre diferentes fotografías, incluidas las tomadas en diferentes puntos de tiempo (es decir, mediciones en serie dentro de un protocolo de estudio). El procedimiento para realizar estas correcciones se detalla en los pasos opcionales 3.1 y 3.2. <ol><li> (Opcional) curva Parcela de registro (exposición) vs log (absorción) utilizando los valores obtenidos de la norma de absorción de escala de grises. </li><li> (Opcional) ajustar las variaciones en el nivel de cada fotografía como sigue: <ol><li> Seleccione F-stop para la lectura (según lo determinado en el paso 1.6) y F-stop para referencia (como se determina en sTEP 1,8). </li><li> Calcular la absorción media de la norma en todas las fotografías en la lectura de F-stop. Este promedio es el estándar de referencia Valor (RSV). </li><li> Calcular la diferencia en la absorción entre la lectura y de referencia configuración F-stop en cada fotografía para el estándar (delta-S) y para cada ROI definido (delta-ROI-1, delta-ROI-2 ... ..delta-ROI-X) </li><li> Calcular la absorción corregido para cada ROI de la siguiente manera: la absorción corregida (ROI-X) = Absorción (ROI-X) en la lectura de F-stop + (RSV - absorción de la norma en la lectura de F-stop) * (delta-ROI-X / delta-S) </li></ol></li><li> Recopilar datos experimentales y realizar análisis de datos utilizando técnicas estadísticas estándar para las variables continuas (es decir, ANOVA, t-test). </li></ol>

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Authors have nothing to disclose.

En este informe una descripción detallada se proporciona de una nueva técnica para la cuantificación de la pérdida de cabello en los roedores. Esta técnica utiliza un generador de imágenes de gel para la adquisición y análisis de imágenes, equipo que está fácilmente disponible en la mayoría de los laboratorios. Las mediciones han demostrado ser robusto a las variaciones menores en la técnica (Figuras 4 - 5), y están bien correlacionados con el grado de pérdida de cabello ...

La alopecia (caída del cabello) puede ser un evento psicológicamente y emocionalmente angustiante con múltiples causas. Calvicie de patrón masculino es la causa más común de alopecia, que afecta a aproximadamente dos tercios de los hombres a los 35 años 1. Un patrón similar de pérdida de cabello se puede observar en las mujeres con síndrome de ovario poliquístico. En ambos de estos trastornos, la pérdida de cabello es de andrógenos mediada. Alopecia también puede ocurrir como una enfermedad autoinmune, alopecia areata, que afecta a 1,7% de la población 2. La alopecia puede ocurrir como efecto secundario de algunos tratamientos médicos, como la quimioterapia 3. Un alto porcentaje (65-85%) de los pacientes de quimioterapia experimentan algún grado de alopecia 4,5. Las consecuencias psicológicas de la pérdida de cabello se han estudiado bien en el entorno de la quimioterapia. Alopecia inducida por la quimioterapia puede provocar ansiedad, depresión, una imagen corporal negativa, baja autoestima y un sentido reducido de bienestar 6,7. Un alto percentaje (47-58%) de los pacientes de cáncer femeninas consideran la pérdida del cabello a ser el aspecto más traumática de la quimioterapia, y hasta 8% de tratamiento de disminución por temor a 4,6 pérdida de cabello. También hay evidencia en la alopecia androgénica para apoyar a la terapia para reducir las consecuencias psicológicas y médicas, incluso de 8,9 pérdida de cabello. Asimismo, se ha informado de la alopecia areata tener graves consecuencias psicológicas 2, y de la naturaleza irregular de la pérdida de cabello puede conducir a un resultado cosmético más desagradable que la mayoría de otras causas de pérdida de cabello. Mientras que las drogas con efectos anti-androgénicos leves (es decir, espironolactona) se habían utilizado con éxito limitado como tratamiento para la alopecia, el primer medicamento eficaz para la alopecia fue minoxidil 10. Este antihipertensivo tiene un efecto secundario observado de causar el crecimiento del cabello, y ahora se utiliza como terapia tópica para muchas formas de alopecia. Sin embargo, las respuestas son a menudo incompletos, con algunos temas mostrando sólo lentaing de pérdida de cabello en lugar de rebrote real 10. Finasteride es un antagonista competitivo de tipo II 5α-reductasa que bloquea la conversión de testosterona a dihidrotestosterona, que resulta en mejoras en la alopecia androgénica, a expensas de bloqueo parcial de andrógenos sistémica. Las tasas de respuesta con a largo plazo (10 años), la terapia son alrededor del 50% 11. En general, a pesar de una considerable investigación en esta área, aún no existe una terapia adecuada para la pérdida del cabello. Durante décadas, los científicos y los médicos han examinado métodos de medición de crecimiento del pelo del cuero cabelludo en ensayos clínicos. Con el desarrollo de medicamentos que tratan la alopecia, ha habido una mayor necesidad de medios fiables, económicas y mínimamente invasivos de medición de crecimiento del cabello y, específicamente, la respuesta a la terapia. La tecnología de análisis de imágenes para una cuantificación precisa de la densidad del pelo en pacientes con trastornos de pérdida de cabello produjeron resultados coherentes y válidos en el pasado usando una variedad de techniquES, incluyendo el análisis de imágenes digitalizadas 12, análisis de imágenes de los pelos individuales y las lesiones de la piel 13, y el escaneo microscópico para cuantificar la masa de pelo del cuero cabelludo en una región definida 14. Desafortunadamente, mientras que las metodologías anteriores han proporcionado una mejor evaluación de la eficacia para el cabello intervenciones en los ensayos clínicos promotora del crecimiento, estos métodos no han sido aplicados a los estudios con roedores en investigaciones preclínicas. Nuestro objetivo es desarrollar un método consistente y fiable para cuantificar la pérdida de cabello en ratones, lo que permitirá a los investigadores a evaluar con mayor precisión y comparar nuevos enfoques terapéuticos para diversas formas de alopecia. Hemos desarrollado una metodología utilizando equipos disponibles en la mayoría de los laboratorios que permitirán la cuantificación rápida y fiable de la densidad del pelo en ratones con pelo marrón o negro. Esta metodología ha sido probada en modelos de ratón de la alopecia inducida por quimioterapia, alopecia areata y la alopecia de waxing. Un protocolo detallado se presenta para realizar estas mediciones, incluidos los datos de validación de C57BL / 6 y C3H / HeJ cepas de ratones. Como esta técnica se basa en la detección de la absorción de luz a partir de pigmentos en el eje del pelo, no puede ser utilizado para detectar el crecimiento del cabello en ratones blancos o ratones albinos.

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">KODAK Gel Logic 100 Imaging System&#160;&#160;</td>
			<td style="width:121px;">Eastman Kodak Company, Rochester, NY, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gel imager for obtaining and analyzing photographs, must have built in light source for reflective photography.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">The Kodak/Tiffen Q-13 Gray Scale</td>
			<td style="width:121px;">Imatest, http://www.imatest.com/docs/q13/</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Greyscale standard</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C57BL/6J Mice</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C3H/HeJ engrafted mouse</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
	</tbody>
</table>

a new technique for quantitative analysis of hair loss in mice using grayscale analysis

La alopecia es una forma común de pérdida de cabello que puede ocurrir en muchas condiciones diferentes, incluyendo la pérdida de cabello de patrón masculino, el síndrome de ovario poliquístico, y la alopecia areata. Alopecia también puede ocurrir como un efecto secundario de la quimioterapia en pacientes con cáncer. En este estudio, nuestro objetivo fue desarrollar un método consistente y fiable para cuantificar la pérdida del pelo en ratones, lo que permitirá a los investigadores a evaluar con precisión y comparar los nuevos enfoques terapéuticos para estas diversas formas de alopecia. El método utiliza un generador de imágenes de gel estándar para obtener y procesar imágenes de los ratones, la medición de la absorción de la luz, que se produce en una proporción aproximada a la cantidad de pelo negro (o gris) en el ratón. Los datos que han sido cuantificados de esta manera se pueden analizar utilizando técnicas estadísticas estándar (es decir, ANOVA, t-test). Esta metodología fue probada en modelos de ratón de la alopecia inducida por quimioterapia, alopecia areata y la alopecia de la depilación. En este informe, el protocolo detallado es presentados para realizar estas mediciones, incluidos los datos de validación de C57BL / 6 y C3H / HeJ cepas de ratones. Esta nueva técnica ofrece una serie de ventajas, incluyendo la relativa simplicidad de aplicación, la confianza en el equipo que está fácilmente disponible en la mayoría de los laboratorios de investigación, y la aplicación de una evaluación objetiva, cuantitativa que es más robusto que las evaluaciones subjetivas. Las mejoras en la cuantificación del crecimiento del pelo en ratones mejorarán estudio de los modelos de alopecia y facilitar la evaluación de nuevas terapias prometedoras en estudios preclínicos.

Esta técnica fue validada utilizando / HeJ injertadas C3H, el modelo de ratón de la alopecia areata 16. Estos animales desarrollan la pérdida mundial de cabello que progresa gradualmente con el tiempo. Sin embargo, la pérdida de cabello se produce en parches, y puede variar de un ratón a la siguiente, la introducción de una variabilidad considerable y obstaculizando evaluaciones cualitativas. Este modelo proporciona una oportunidad para poner a prueba las correlaciones entre las mediciones de densidad ?...

Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Una nueva técnica de análisis cuantitativos de la pérdida del pelo en ratones Uso del análisis de escala de grises

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

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8.9K vistas.  Children's Hospital at Montefiore. Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Video: A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Investigación

Biología

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Células vivas, imágenes y análisis cuantitativo de las células epiteliales embrionarias en Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Un cabello no invasiva técnica de muestreo para obtener ADN de alta calidad de esquiva Pequeños Mamíferos

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Cuantitativa de células vivas por fluorescencia microscopia análisis de la levadura de fisión

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Análisis cuantitativo de la migración desordenada de las células mediante microscopía de Time-lapse vídeo

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Un flujo de trabajo de Análisis Cuantitativo Gimnasio

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Análisis cuantitativo de la autofagia mediante microscopía de fluorescencia avanzada 3D

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

Extracción y análisis de cortisol de Derechos Humanos y de mono de pelo

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...