Trabajo descrito en este manuscrito fue posible gracias al financiamiento de la Fundación Patrick F. Taylor.

1. Cromosoma La recolección de células adherentes <ol><li> Protocolo Estándar <ol><li> Se cultivan las células de acuerdo a condiciones específicas de cultivo celular. Cuando las células han alcanzado la fase logarítmica (80% de confluencia), añadir 10 l / ml de Colcemid al matraz de cultivo de células. Se recomienda un mínimo de 2 x 10 6 células. </li><li> Se incuban las células a 37 º C en un incubador de CO2 al 5% durante 45 min. Usando una pipeta estéril, transferir los medios de las células en un tubo cónico de 15 ml. Ponga a un lado. </li><li> Lave suavemente las células mediante la adición de 2 ml de HBSS Buffer en el matraz. Amortiguar remolino y luego retire con una pipeta. Descartar. </li><li> Añadir 1 ml de tripsina, asegurándose de que cubre toda la superficie del matraz. Sólo dejar las células en tripsina durante aproximadamente 2 min. Una vez que la mayoría de las células se han separado, pipetear los medios de comunicación en el tubo cónico de nuevo en las células. </li><li> Transferir la suspensión celular en alícuotas de 10 ml en tubos de 15 ml cónicos.Centrifugar a 200 xg durante 10 min. Aspirar el sobrenadante y resuspender el precipitado. </li><li> Añadir 10 ml de 0,075 M de KCl que ha sido precalentada a 37 º C al sedimento que queda en el tubo cónico. Tubo Vortex a velocidad media para mezclar KCl y células. </li><li> Se incuban las células a 37 ° C durante 10 min. Centrifugar a 200 xg durante 5 min a 25 ° C. Aspirar el sobrenadante (hasta unos 0,5 ml restos) y pellet se resuspende. </li><li> Añadir cuidadosamente 5 ml de fijador fresco de Carnoy (3:1 de metanol: ácido acético glacial) a las células, mientras que vórtex. Añadir a continuación 5 ml más de fijador sin agitación durante un total de 10 ml. </li><li> Centrifugar a 200 xg durante 5 min. Retire las células sobrenadante y volver a suspender. Añadir 5 ml de fijador a cada tubo. </li><li> Centrifugar a 200 xg durante 5 min. Retire las células sobrenadante y volver a suspender. Añadir 5 ml de fijador a cada tubo. Las células se pueden almacenar en 4 ° C durante hasta un año. </li></ol></li><li> Modificación al protocolo: harvesti cromosómicasng de líneas celulares linfoblastoides <ol><li> Se cultivan las células de acuerdo a condiciones específicas de cultivo celular. (Use matraz apropiado para la cantidad de células que se cultiva). Cuando las células han alcanzado la fase logarítmica (aproximadamente 2 días después de que las células se dividen), añadir 10 l / ml de Colcemid al matraz de cultivo de células. </li></ol></li><li> Modificación al protocolo: Cromosoma recolección de la sangre entera Fitohemaglutinina (PHA), una lectina derivada del frijol rojo, es un mitógeno potente para las células T humanas 16. 72 h después de la adición de PHA a la cultura, aproximadamente el 45% de las células están en la fase S. Esto representa el pico de actividad mitótica, y es el punto óptimo para la cosecha de estudios cromosómicos. Otros mitógenos tales como hierba carmín (1-10 g / ml) se pueden utilizar en el análisis de células B 17,18. <ol><li> Recoge al menos 1 ml de sangre completa en un tubo de heparina sódica superior verde. Utilice dentro de 3 días después de la recogida. Sangre tienda a temperatura ambiente sinhasta listo para usar. </li><li> Alícuota de 0,25 ml de sangre completa en 10 ml de medio RPMI completo que contiene L-glutamina (suero bovino fetal al 20%, 1% penicilina / estreptomicina, 1% de fungizona, y 1% de PHA). Cultivo a 37 ° C con 5% de CO 2. </li></ol></li><li> Modificación al protocolo: Cromosoma recolección de la médula ósea El análisis citogenético de la médula ósea es útil en muchos trastornos hematológicas malignas como la observación de un clon cromosómicamente anormal puede ser diagnóstico para un tipo específico de leucemia. También se utilizan estudios cromosómicos para evaluar la progresión de la enfermedad, tales como la aparición de crisis blástica y respuesta al tratamiento. Dado que las células de la médula ósea se están dividiendo activamente, sin estimulación por mitógenos es necesario 9,19. Para las leucemias agudas culturas 24 y 48 h también se establezcan. <ol><li> Recoge al menos 1 ml de médula ósea en un tubo con heparina de sodio superior verde. Alícuota de 0,25 ml de médula ósea en 10 ml de medio RPMI completo que contiene L-glutamina (20% de suero fetal bovino, 1% penicilina / estreptomicina, 1% de fungizona). Cultivo a 37 ° C con 5% de CO 2. </li></ol></li></ol> 2. Slide Preparación y tinción sólida Una evaluación rápida de una diapositiva representante le proporcionará información sobre la calidad de la cosecha antes de continuar con otros procedimientos tales como bandas G, FISH, CGH, o SKY. Algunos laboratorios prefieren tinción sólida de las células para la recolección rápida y evaluación cromosoma. Alternativamente, un microscopio de contraste de fase puede ser utilizado para este análisis. Los portaobjetos se preparan mejor cuando la humedad es de aproximadamente 50% y la temperatura ambiente (20-25 ° C). <ol><li> Centrifugar las células a 200 xg durante 5 min a 25 ° C. Eliminar el sobrenadante hasta que sólo 0,3-0,5 ml restos. </li><li> Después de resuspender suavemente el sedimento, de pipeta tres gotas de la suspensión de células a partir de una distancia de aproximadamente 2 en sobre un portaobjetos que está inclinado en un ángulo de aproximadamente 45 ° y permitir que la suspensiónrodar a través de la diapositiva. Añada una gran gota de fijador de Carnoy fresco a la diapositiva. </li><li> Seque la parte posterior de la diapositiva en una toalla de papel y luego sentarse la diapositiva a secar durante al menos 10 min. La diapositiva debe estar completamente seco. </li><li> Prepare fresco tinción Giemsa solución (3:1 proporción de Gurr Buffer y Giemsa). Colocar los portaobjetos en una rejilla de tinción. Cubra toda la diapositiva en la solución de tinción Giemsa. Deje que las diapositivas permanecen en la solución de tinción durante 5 min. Enjuagar los portaobjetos con agua destilada, dejar escurrir y dejar secar al aire. </li><li> Añadir 4 gotas de Permount y una hoja de cubierta de la diapositiva. Asegúrese de que no hay burbujas bajo el cubreobjetos. El exceso Permount se puede quitar con una toalla de papel. </li><li> Analice las células con un microscopio de luz con una lupa de 10X y 100X. Si las células en metafase son abundantes y bien propagación, las diapositivas restantes pueden utilizarse para otros procedimientos experimentales. </li></ol> 3. G-Banding utilizando tripsina y Giemsa (GTG) &lt;p&gt; La tripsina es una enzima proteolítica que desnaturaliza las histonas euchromatic en regiones de ADN con una mayor actividad transcripcional resultante. Después de la tinción de Giemsa, estas regiones aparecen como bandas de luz. Cromatina altamente condensado con poca o ninguna actividad transcripcional (heterocromatina) tendrá una gran parte de sus histonas protegidas de la tripsina y por lo tanto se mancha oscura después de la tinción de Giemsa. Es esencial inicialmente G-banda una diapositiva a supervisar las condiciones y ajuste la sincronización de la tripsina en caso de necesidad para las diapositivas siguientes. <ol><li> Añadir las siguientes soluciones para 4 jarras Coplin. Tarro # 1 = 30 ml de HBSS 1X y 4 ml de 10x tripsina (0,5%) Jar # 2 = 50 ml de 1x HBSS Tarro # 3 = 45 ml de HBSS 1X y 5 ml de suero fetal bovino Jar # 4 = 50 ml de 1x HBSS </li><li> Sumerja cada diapositiva de Jar # 1 durante 5 segundos, enjuague rápido en Jar # 2, deje cada diapositiva de Jar # 3 durante al menos 30 segundos, enjuague rápido en Jar # 4 luego permitir que se sequen. </li><li> Preparar fresh Giemsa tinción Solución (relación 3:1 de Gurr Buffer y Giemsa). Colocar los portaobjetos en una rejilla de tinción. Cubra toda la diapositiva en la solución de tinción Giemsa. Deje que las diapositivas permanecen en la solución de tinción durante 5 min. </li><li> Enjuagar los portaobjetos en agua destilada en el mismo orden en que estaban manchadas. Deje que los portaobjetos se sequen durante unos 10 min. La diapositiva debe estar completamente seco. </li><li> Añadir 4 gotas de Permount y una hoja de cubierta de la diapositiva. Asegúrese de que no hay burbujas bajo el cubreobjetos. El exceso Permount se puede quitar con una toalla de papel. </li><li> Analice las células con un microscopio de luz con una lupa de 10X y 100X. Ajuste de temporización tripsina de el resto de las diapositivas en base a los resultados de la primera diapositiva. </li></ol>

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Hemos utilizado el presente procedimiento para el cromosoma aislamiento a partir de células de diversos organismos, incluyendo diversos tipos de células obtenidos a partir de humano, macacos Rhesus, ratas, y ratones 11,20-22. Se proporciona el protocolo estándar, pero ciertos pasos clave y variables puede ser necesario ajustar para estos diversos tipos de células. Varios pasos específicos son cruciales tanto en la preparación cromosómica y bandas G para asegurar la mejor calidad posible de los resultad...

El análisis cromosómico es una técnica convencional utilizada en todo el mundo para el diagnóstico de la inestabilidad cromosómica y reordenamientos que conducen a trastornos genéticos y 1,2,8,9 malignidad. Además, una resolución más alta para el diagnóstico y la investigación de anomalías genéticas constitucionales y el cáncer adquirida se puede lograr con la combinación de los procedimientos citogenéticos clásicos y metodologías de citogenética molecular, tales como hibridación in situ fluorescente (FISH), hibridación genómica comparativa (CGH) y cariotipo espectral (SKY) 14,15. Más recientemente, estas técnicas se han utilizado para la evaluación de la inestabilidad cromosómica asociada con la investigación de células madre. Anomalías del cariotipo como aneuploidía de células a largo plazo cultivadas embrionarias (ES) y las células madre adultas de diversos organismos han sido reportados por varios laboratorios. Evidencia reciente apoya que algunas líneas celulares son inherentemente más inclinados al cromosoma instabilidad, independientemente de las condiciones de cultivo. Por esta razón, cuando el establecimiento y / o mantenimiento de líneas de células madre rhesus humanos, de ratón, o bien, se recomienda el análisis de cromosomas, como parte del proceso de control de calidad. Muchos informes describen el creciente interés en el uso de la citogenética de rutina y moleculares para vigilar la estabilidad cromosómica de las células madre y células malignas o varios organismos en cultivo 10-13. Estos protocolos son cada vez más utilizados por los laboratorios no genéticos para la evaluación cromosómica rápida de sus células cultivadas 13. Presentamos nuestros procedimientos básicos para la preparación de cromosomas de diversos tipos de células, que se pueden aplicar para ambos propósitos y células clínicos y de investigación derivadas de diversos organismos.

<table border="0" cellpadding="0" cellspacing="0" width="662">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">KaryoMAX Colcemid</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:145px;">15212-012</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS Buffer</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin EDTA 10x</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer Tablets &#34;GURR&#34;</td>
			<td>Gibco</td>
			<td>10580-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Geimsa Stain</td>
			<td>Ricca Chemical Company</td>
			<td>3250-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>6700130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromosome preparation from cultured cells

Cromosoma (citogenética) análisis se utiliza ampliamente para la detección de la inestabilidad cromosómica. Cuando se sigue por bandeo G y las técnicas moleculares como la hibridación in situ fluorescente (FISH), este ensayo tiene la extraordinaria capacidad de analizar las células individuales de las aberraciones que implican ganancias o pérdidas de partes del genoma y reordenamientos que afecte a uno o más cromosomas. En los seres humanos, las anomalías cromosómicas ocurren en aproximadamente 1 de cada 160 nacidos vivos a 1,2, el 60-80% de todos los abortos involuntarios 3,4, 10% de los mortinatos 2,5, 13% de los individuos con enfermedad cardíaca congénita 6, 3-6% de los casos de infertilidad 2, y en muchos pacientes con retraso en el desarrollo y defectos de nacimiento 7. El análisis citogenético de malignidad se utiliza de forma rutinaria por los investigadores y los clínicos, ya que se ha demostrado que las observaciones de anomalías cromosómicas clonales tener tanto significado diagnóstico y pronóstico 8,9.60, el aislamiento del cromosoma tiene un valor incalculable para la terapia génica y la investigación con células madre de organismos incluyendo primates no humanos y roedores 10-13. Los cromosomas se pueden aislar a partir de células de tejidos vivos, incluyendo linfocitos de sangre, fibroblastos de la piel, amniocitos, placenta, médula ósea, y las muestras de tumor. Los cromosomas son analizados en la etapa de metafase de la mitosis, cuando más se condensan y por lo tanto más claramente visible. El primer paso de la técnica cromosoma aislamiento implica la interrupción de las fibras del huso por incubación con Colcemid, para evitar que las células de proceder a la etapa de la anafase posterior. Las células se trataron luego con una solución hipotónica y se conservan en su estado hinchado con fijador de Carnoy. Las células son luego caer sobre portaobjetos y luego se pueden utilizar para una variedad de procedimientos. Bandeo G consiste en el tratamiento de tripsina seguido de tinción con Giemsa para crear la luz característica y bandas oscuras. Lo mismo procedure para aislar cromosomas se puede utilizar para la preparación de células para los procedimientos tales como la hibridación in situ fluorescente (FISH), hibridación genómica comparativa (CGH), y cariotipo espectral (SKY) 14,15.

Metafase para untar de alta calidad son esenciales para el análisis cromosómico. Un ensayo exitoso produce cromosomas que están bien repartidas y de la morfología cromosómica adecuada. Cromosomas correctamente bandas-G contienen la luz característica y los patrones de bandas oscuras. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> Figura 1. ...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

Los cromosomas se pueden aislar de las células vivas, tales como linfocitos o fibroblastos de la piel, y a partir de organismos, incluyendo seres humanos o ratones. Estas preparaciones cromosómicas se pueden utilizar más de rutina bandeo G y procedimientos citogenéticos moleculares como la hibridación fluorescente in situ (FISH), la hibridación genómica comparada (CGH) y cariotipo espectral (SKY).

Cromosoma Preparación de las células cultivadas

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

chromosome-preparation-from-cultured-cells

Research

JoVE Journal

Biology

80.7K vistas.  Louisiana State University Health Science Center.  Los cromosomas se pueden aislar de las células vivas, tales como linfocitos o fibroblastos de la piel, y a partir de organismos, incluyendo seres humanos o ratones. Estas preparaciones cromosómicas se pueden utilizar más de rutina bandeo G y procedimientos citogenéticos moleculares como la hibridación fluorescente in situ (FISH), la hibridación genómica comparada (CGH) y cariotipo espectral (SKY). 

Video: Chromosome Preparation From Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Contar y determinar la viabilidad de las células cultivadas

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Investigación

Biología

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment.
The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies.
Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidham's method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.

La preparación de bloques de células a partir de muestras de citología con predominio de células aisladas individualmente

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Preparación de los cromosomas politénicos de Drosophila Calabazas para el Etiquetado de anticuerpos

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basado en la fluorescencia de medición de la entrada de calcio de la tienda que funciona en las células vivas: a partir de células cancerosas cultivadas de fibra muscular esquelética

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualización del retículo endoplasmático subdominios en células cultivadas

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

Ensayos Reporteros Massively Parallel en células de mamífero cultivadas

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Aislamiento y análisis funcional de las mitocondrias de las células cultivadas y ratón de Tejidos

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

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, ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Una caída Cromosoma Aire seco Rápido Método de preparación adecuados para FISH en Plantas

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Preparación del cromosoma meiótico se extiende desde ratón espermatocitos

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...