Nos gustaría dar las gracias a Marc Leonard de Carleton Immersive Media Studio expertos para la asistencia técnica en la producción y edición de video y el Dr. Martin Bertrand de Carleton Immersive Media Studio / Laboratorio de Regeneración Neural de asistencia modelo visual. Los autores agradecen el asesoramiento de la Dra. Marilyn Keaney y todo su personal especializado en la Universidad de Ottawa y Cuidado de Animales Servicios Veterinarios. Este trabajo fue financiado por el Instituto Canadiense de Investigación en Salud (CIHR, RP 62826) para SALB, el Instituto de Envejecimiento y CIHR Iniciativa para la Formación Estratégica de Investigación de la Salud / Programa de Formación en CIHR lipidomics Neurodegenerativas (TGF 96121) para SALB y SF, Fundación Canadiense de Innovación a SF, Ontario Innovation Trust a SF, y Autodesk investigación a la ciencia ficción. ACM recibe un CIHR Banting y Best premio doctoral. NV recibe una beca post-profesional del Instituto de Mayores y el Programa de Capacitación en CIHR lipidomics neurodegenerativas.

1. Preparación de Reactivos <ol><li> Para el lavado vaginal estéril, autoclave agua doblemente destilada (DDH 2 O) y guárdela en un recipiente herméticamente cerrado, a temperatura ambiente hasta que se necesite. </li><li> Para la evaluación citológica, añadir 0,1 g de polvo de cristal violeta a 100 ml de ddH 2 O. Mezclar bien. Tinción de cristal violeta (0,1%) se puede almacenar en un recipiente herméticamente cerrado a temperatura ambiente hasta que se necesite. </li></ol> 2. Recolección de células vaginales (lavado vaginal) <ol><li> Colocar una bombilla de látex en el extremo de una punta estéril 200 l y elaborar aproximadamente 100 l de ddH 2 O estéril utilizando las gradaciones en la punta como una guía volumen. </li><li> Levante el ratón fuera de su jaula y coloque la jaula en la tolva (tapa) con su extremo trasero / trasero hacia usted. </li><li> Sujete firmemente la cola y elevar el extremo posterior. El ratón tendrá ahora sólo sus patas delanteras prensiles la tolva. En este punto, el ratón puede orinar. Si es así, Espere hasta que deje de orinar. ¿Debería haber orina que queda en la entrada del canal vaginal, es posible que desee aclarar la abertura con el exceso de O 2 ddH usando una punta diferente (es decir, no con la punta de recogida de muestras). </li><li> Coloque el extremo de la ddH 2 O llena de punta en la abertura del canal vaginal, teniendo cuidado de no penetrar en el orificio vaginal como (y cervical) estimulación puede inducir pseudopreñez en ratas 1,2. Los informes recientes sugieren que los ratones son menos susceptibles a este efecto, sin embargo se debe tener cuidado para reducir al mínimo el grado de invasividad en análisis repetidos 3. </li><li> Presione suavemente la bombilla para expulsar un cuarto a la mitad del volumen de agua (~ 25-50 l) en la apertura del canal vaginal. El líquido aspirado de forma espontánea en el canal sin la punta de inserción. Lentamente libere la presión ejercida sobre el bulbo. El fluido se retire de nuevo en la punta. Evite liberación de presión demasiado rápido para evitar la aspiración de líquidoen el bulbo. Una punta filtrada puede ser útil para este propósito. </li><li> Repetir los tiempos de las etapas anteriores 4-5 utilizando la misma punta, bulbo, y el líquido para obtener un número suficiente de células en una sola muestra. </li><li> Coloque el fluido sobre portaobjetos de vidrio, y permitir que el frotis se seque por completo a temperatura ambiente. Una vez seca, estos frotis estrales puede teñir inmediatamente o almacenarse y se tiñeron en una fecha posterior. </li></ol> 3. Tinción citológica utilizando Crystal Violet * 4 <ol><li> Colocar el portaobjetos en seco en un vaso de Coplin (u otro recipiente de tinción comparable) que contiene el colorante cristal violeta durante 1 min. </li><li> Retirar a un segundo vaso de Coplin que contiene ddH 2 O. Lave el portaobjetos con ddH 2 O durante 1 min. Repetir. </li><li> Retire el exceso ddH 2 O de los bordes de la diapositiva con un limpiador de tejidos ligeros, evitando el contacto con la tinción. </li><li> Pipetear aproximadamente 15 l de glicerol en la parte superior de la tinción y la taparesbale. Alternativamente, otros reactivos histológicos de montaje pueden ser utilizados para obtener un más permanente, no difusora mancha. </li></ol> * El método de tinción descrito aquí es el procedimiento más simple que puede realizarse en cualquier laboratorio. Otros métodos pueden proporcionar detalles adicionales. Por ejemplo, mediante la tinción de Papanicolaou, la madurez de las células epiteliales nucleadas se pueden distinguir con células menos maduras maduras células teñidas de color turquesa y más rosa o anaranjado-teñidas. Estas diferencias se pueden utilizar para la etapa proestro temprano o tardío. 4 4. Citología Vaginal <ol><li> Examinar el frotis al microscopio óptico para determinar los tipos de células presentes. El examen microscópico se debe hacer inmediatamente después de la tinción de cristal violeta como se difundirá a partir de las células con el tiempo cuando se usa glicerol para cubreobjetos. Las microfotografías deben tomarse en el momento de análisis para documentar la citología. </li><li> Comience por examinar la entire frotis con un aumento menor. Seleccione un área representativa y pasar a un mayor aumento. Verá cornificación células epiteliales escamosas, leucocitos, y / o células epiteliales nucleadas (resultados representativos, la figura 1A-C). La proporción de células presentes le permitirá determinar la etapa de estro de su ratón en el momento de la toma de muestras (resultados representativos, Figura G-1D) y su estado hormonal inmediata (discusión, Figura 2). </li></ol> 5. Los resultados representativos Citología: Tres tipos de células primarias pueden ser detectados en muestras de frotis vaginales: (1) células nucleadas epiteliales (Figura 1A), (2) cornificación células epiteliales escamosas (Figura 1B), y (3) los leucocitos (Figura 1C). Nucleados células epiteliales tienen un citoplasma ligeramente manchado, manchado oscuro membrana plasmática, y un núcleo oval ( <strong&gt; Figura 1A). Cornified células epiteliales escamosas son uniformemente manchado, más de forma poligonal que sus predecesores epiteliales nucleadas, y carecen de un núcleo (Figura 1B). Los leucocitos polimorfonucleares se pueden distinguir de las células epiteliales por su forma irregular, núcleos polimórfico oscuro manchado, y el pequeño tamaño (Figura 1C, las flechas negras). En caso de contaminación de la orina estar presente en el frotis, cristales de ácido úrico se detectan fácilmente por sus estructuras cristalinas diferentes a cualquier tipo de celda esperados (Figura 3). Si esto ocurre, y la detección oscura de tipo celular predominante, la prueba debe ser desechado y no se utiliza para fines de estadificación. Montaje: La proporción relativa de los tipos de células observadas en frotis se puede utilizar para identificar la fase del ciclo estral de su ratón en el día de la recogida de muestras (Figura 1D-G). Durante el proestro, las células son casi exclusivamenteracimos de vuelta, bien formado nucleados células epiteliales (Figura 1D, célula representativa indicada por la flecha blanca). Durante el estro, las células son predominantemente cornificación células epiteliales escamosas, presentes en racimos compactados (Figura 1E, célula representante indicado por punta de flecha). Durante metaestro, pequeñas leucocitos oscuramente teñidos predominan (Figura 1F, célula representativa indicada por la flecha negro). Cornified células epiteliales escamosas se puede observar, a menudo en fragmentos, (Figura 1F, célula representativa indicada por negro punta de flecha). Durante el diestro, raras cornificadas células epiteliales escamosas puede aún estar presente (Figura 1G, célula representativa indicada por negro punta de flecha), sin embargo aún predominan los leucocitos (Figura 1G, célula representativa indicada por la flecha negro). Metaestro puede distinguirse de diestro por la aparición de células epiteliales nucleadas en diestro ( <stron<html> g&gt; Figura 1G, célula representativa indicada por la flecha blanca). <img src="/files/ftp_upload/4389/4389fig1.jpg" alt="Figura 1" /> Figura 1. Evaluación citológico de frotis vaginal puede ser utilizado para identificar la etapa estral tres principales tipos de células se detectan en muestras de frotis vaginales:. (A) las células epiteliales nucleadas, (B) cornificadas células epiteliales escamosas, y leucocitos (C). La proporción de estos tipos de células presentes en el frotis se puede utilizar para identificar en ratones (D) proestro, (E) estro, (F) metaestro, o diestro (G) como se describe en los resultados representativos. Puntas de flecha negras en E, F y G a punto representativos cornificadas células epiteliales escamosas. Las flechas negras en C, F y G a punto leykocytes representativas. Las flechas blancas en D y G representante más destacado nucleados células epiteliales. . Jpg &quot;alt =&quot; Figura 2 &quot;/&gt; Figura 2. Citología vaginal smear refleja hechos subyacentes endocrinos. Detalles se proporcionan también en discusión. <a href="http://www.jove.com/files/ftp_upload/4389/4389fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img src="/files/ftp_upload/4389/4389fig3.jpg" alt="Figura 3" /> Figura 3. Cristales de ácido úrico puede estar presente siguiente de tinción de cristal violeta de orina contaminados-muestras. (A) los cristales son transparentes y pueden ser de varios tamaños (flechas y la región en caja magnificados en (B)). No hay células están presentes en este campo. En caso de contaminación úrico cristales de ácido dentro de los campos utilizados para la tinción citológica estar presente, puede ser difícil identificar con precisión los tipos de células presentes y el frotis debe ser desechado. Las barras de escala = 50 micras.

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Los autores declaran no tener ningún conflicto de intereses. Todos los experimentos con animales se llevaron a cabo en estricta conformidad con las normas y reglamentos establecidos por la Universidad de Ottawa Animal Care Comité y el Consejo Canadiense de Protección de los Animales.

Estos cambios en la tipología de células son indicativos de eventos endocrinos subyacentes. La fase proestro del ciclo estral se corresponde con la fase folicular humano del ciclo menstrual y 5 se define por un aumento preovulatorio en circulante 17-β-estradiol niveles 6, así como un aumento pequeño en la prolactina 7 (Figura 2, proestro, panel izquierdo). El aumento de 17-β-estradiol estimula indirectamente liberadora de gonadotropina hormona de neuronas en el hipotálamo y el septo que, a su vez, activan las células sensibles en la pituitaria anterior para liberar hormona luteinizante y hormona estimulante del folículo en la circulación 8,9 (Figura 2 , Proestro, panel izquierdo). En los frotis vaginales obtenidas de los animales en proestro, las células son casi exclusivamente ovales células nucleadas epiteliales (Figura 1D, la figura 2, el proestro, panel derecho). El pico en horm estimulante del folículouna ovulación y los niveles de las señales de entrada en estro 10,11. Durante el estro, 17-β-estradiol disminuyen los niveles de los niveles de prolactina y pico de 6,7 (Figura 2, estro, panel izquierdo). Frotis vaginales se caracterizan por detección casi exclusivo de forma irregular cornificadas células epiteliales escamosas a menudo en grupos (Figura 1E, la Figura 2, el estro, panel derecho). Entrada en metaestro coincide con un aumento continuo en los niveles de hormonas progesterona y 6 se corresponde con el inicio de la fase lútea humano 12 (Figura 2, metaestro, panel izquierdo). Como los niveles de progesterona comienzan a aumentar y hay un aumento pequeño en el 17-β-estradiol los niveles en respuesta a la activación del cuerpo lúteo 6,13,14 (Figura 2, metaestro, panel izquierdo). Los tipos de células presentes en los frotis vaginales durante esta etapa son células fragmentadas, cornificadas epiteliales y leucocitos más pequeñas más oscuras manchadas (Figure 1F, la figura 2, metaestro, panel derecho). Por último, la entrada en el diestro en ratones se produce y los niveles circulantes de progesterona pico 6, correspondiente a la fase lútea tardía humano 12. Regresión del cuerpo lúteo conduce a una posterior disminución brusca en los niveles de progesterona 15,16 (Figura 2, Diestro, panel izquierdo). Los leucocitos predominan en los frotis durante el diestro. La frecuencia de células epiteliales cornificadas y nucleadas se reduce células epiteliales empiezan a ser detectados justo antes de la transición a la proestro (Figura 1G, la Figura 2, diestro, panel derecho). En resumen, este protocolo simple rutina puede ser utilizada para estimar las fluctuaciones hormonales diarias y establecer etapa estral en ratones experimentales sin alterar el estado reproductivo si las toman las siguientes precauciones. El muestreo se debe realizar no más de una vez al día usando el protocolo no invasivo described aquí, en comparación con la penetración repetida de la cavidad vaginal, la aspiración y la agitación. Esto puede causar irritación vaginal resulta en una 17 respuesta inflamatoria resultante en leucocitos y otros tipos de células para estar presentes en los frotis que pueden confundir la evaluación citológica. Además, incluso en colonias alojadas las mujeres, es normal ver a diestro prolongados y fases de estro en ratones diferentes como la inducción y de anestro 18 y esta identificación es útil en la interpretación de impacto hormonal en la salud reproductiva, género y estudios de enfermedades. La variabilidad en la duración del ciclo también se introduce con la edad y por las diferencias de vivienda en colonias (individual o en grupo de viviendas-) de las hembras 7,19-21. Hembras alojadas en femenino-colonias sólo puede dejar la bicicleta y entrar en un estado de prolongada diestro 18,22,23 aunque la bicicleta puede ser restablecida por la exposición a las jaulas pretratados con orina masculino para obtener 24,25 bicicleta. Por lo tanto, para establecer individual longitudes de ciclo para un ratón dado, se recomienda que las evaluaciones no invasivos descritos aquí puede realizar diariamente, con cuidado, hasta dos ciclos completos se observan.

<table border="1"><tbody><tr><td> Nombre </td><td> Empresa </td><td> Catálogo # </td></tr><tr><td> Puntas de pipeta estériles 200 mu l </td><td> DiaMed </td><td> E340901 </td></tr><tr><td> Pera de látex (1 ml) </td><td> Pescador </td><td> 03-488-21 </td></tr><tr><td> Portaobjetos de vidrio </td><td> Pescador </td><td> 12-550-15 </td></tr><tr><td> Crystal Violet mancha (25 g) </td><td> Pescador </td><td> C581-25 </td></tr><tr><td> Trabajo liviano Limpiaparabrisas tejido </td><td> VWR </td><td> 82003-820 </td></tr><tr><td> Glicerol </td><td> Pescador </td><td> BP229-1 </td></tr><tr><td> Microscopio Glass Cover (22x30) </td><td> Pescador </td><td> 12-544A </td></tr></tbody></table>

performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification

Un medio rápido para evaluar el estado reproductivo en roedores es útil no sólo en el estudio de la disfunción reproductiva, sino que también es necesario para la producción de nuevos modelos de ratón de la enfermedad y las investigaciones en la regulación hormonal de la degeneración del tejido (o regeneración) después de la estimulación patológica. El murino reproductiva (o estro) ciclo se divide en 4 etapas: proestro, estro, metaestro y diestro. Fluctuaciones definidas en los niveles circulantes de los esteroides ováricos 17-β-estradiol y progesterona, las gonadotropinas luteinizante y hormonas estimulantes del folículo, y la prolactina luteotropic hormona transición de la señal a través de estas etapas de reproducción. Los cambios en la tipología de células dentro del canal vaginal murino reflejar estos eventos endocrinos subyacentes. Evaluación diaria de la proporción relativa de células nucleadas cornificadas epiteliales, células epiteliales escamosas, y leucocitos presentes en los frotis vaginales se pueden utilizar para identificar murino estraletapas. El grado de invasividad, sin embargo, empleado en la recogida de estas muestras puede alterar el estado reproductivo y provocar una respuesta inflamatoria que puede confundir la evaluación citológico de frotis. Aquí se describe un simple, no invasivo protocolo que se puede utilizar para determinar el estadio del ciclo estral de una hembra de ratón sin alterar su ciclo reproductivo. Detallamos cómo diferenciar entre las cuatro fases del ciclo estral por la recolección y el análisis de la tipología predominante de células en frotis vaginales y nos muestran cómo estos cambios pueden ser interpretados con respecto al estado endocrino.

Watch this Scientific Journal Video about Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification at JoVE.com

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

Aquí se describe cómo identificar la etapa del ratón reproductiva (proestro, estro, metaestro o diestro) por simple, no invasivo recogida y evaluación citológica de muestras de frotis vaginales. Nos describen con más detalle cómo citología vaginal refleja los niveles circulantes de hormonas subyacentes transición a través del ciclo reproductivo murino.

Realizar el lavado de la vagina, tinción de cristal violeta y Evaluación citológica vaginal para ratón Celo identificación Staging Ciclo

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

performing-vaginal-lavage-crystal-violet-staining-vaginal-cytological

Research

JoVE Journal

Biology

110.1K vistas.  Neural Regeneration Laboratory and Ottawa Institute of Systems Biology. Aquí se describe cómo identificar la etapa del ratón reproductiva (proestro, estro, metaestro o diestro) por simple, no invasivo recogida y evaluación citológica de muestras de frotis vaginales. Nos describen con más detalle cómo citología vaginal refleja los niveles circulantes de hormonas subyacentes transición a través del ciclo reproductivo murino.

Video: Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

A reversible, método no invasivo para las mediciones de resistencia de las vías y Toma de Muestras Líquido del Lavado broncoalveolar en ratones

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Investigación

Biología

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Evaluación de la glándula mamaria de desarrollo y función en modelos de ratón

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

Teléfono directo Análisis de Ciclo de<em&gt; Drosophila</em&gt; Los tejidos que usan la acústica Attune Centrándose citómetro y Vybrant DyeCycle ADN Violet Stain

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing para la identificación microbiana y caracterización

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Análisis citológico de espermatogénesis: Los preparativos en directo y fijas de<em&gt; Drosophila</em&gt; Testículos

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

Métodos para pupa períodos de prueba y medición de pigmentación de ala de Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno celular masiva inyección de células madre embrionarias conduce a mayores tasas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Cuantificación de lípidos abundancia y evaluación de la distribución de lípidos en Caenorhabditis elegans por Nilo rojo y aceite rojo O manchas

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Identificación y disección de diversos depósitos de adipose de ratón

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...