Reconocemos Juliann Jaumotte a la idea de ahorrar en los volúmenes de reactivos en el ensayo de ATP. Estamos profundamente agradecidos por el apoyo administrativo extraordinario de María Caruso, Deb Willson, y Jackie Farrer y para la Escuela de Farmacia de Mylan para proporcionar apoyo financiero para estos estudios. Gracias también a las Enfermedades Hunkele Temido Fundación y el Parkinson y Trastornos del Movimiento de la Fundación por su apoyo financiero de los estudios primarios de neuronas.

Ninguno de los autores tiene ningún conflicto a revelar.

Hemos encontrado que la intensidad de la señal en los tres ensayos de viabilidad es lineal y se correlacionó con densidad de placas. Sin embargo, no todos los ensayos son igualmente sensibles a 2 veces o cambios 1.5 veces en la densidad de placas. Para las células N2a, los ensayos de infrarrojos son menos sensibles que el ensayo de ATP, particularmente a densidades más bajas de chapado. Aunque los ensayos de infrarrojos son menos sensibles que los ATP, los ensayos DRAQ5 + Zafiro y los ensayos de α-tubulina están e...

El ensayo de viabilidad más común en las ciencias biológicas implica el recuento de células. Esto se evidencia por un análisis de los mejores (los más recientes) 200 publicaciones que aparecieron en PubMed con cualquiera de las palabras clave &quot;in vitro&quot; o &quot;cultura&quot; en 29/04/2013 y 30/04/2013. De estas publicaciones, el 23,5% utilizan ensayos de recuento de células, incluyendo recuento de número de células manuales, el número de células automatizado cuenta con software de imágenes, y Trypan exclusión de azul. El ensayo Live / Dead se utilizó en el 1% de estas publicaciones. El número de publicaciones utilizando el MTT (3 - (4,5-dimetiltiazol-2-il) -2,5-difeniltetrazolio) de ensayo para la viabilidad metabólica era 11%. Esta revisión de la literatura también muestra que el número de publicaciones que utilizan ensayos tales como MTT junto con ensayos de recuento de células fue sólo del 3,5%. A pesar de la tendencia a usar un ensayo de viabilidad por sí mismo, la evaluación de la función celular en combinación con el número de células parece ser la mejor elección para la evaluación de i celularntegridad. Los recuentos de células por sí mismas no son suficientes debido a que las células restantes pueden no ser funcional o sano a pesar de que están presentes en el pocillo 1,2. A la inversa, las medidas funcionales tales como ATP pueden aumentar o disminuir en ausencia de cambios paralelos en el número de células. El desacoplamiento de las lecturas metabólicas del número de células sugiere que los ensayos de la ATP y MTT nunca debe ser usado como el único ensayo de viabilidad. En el presente informe, tres ensayos de viabilidad control que analicen tanto las estructuras celulares y la función metabólica se describen, por una visión más completa de la integridad celular que cualquier ensayo por sí solo puede permitirse. Dos de nuestros ensayos requieren una cámara infrarroja que mide la fluorescencia en los 700 y 800 nm canales. El ruido es baja en las longitudes de onda infrarrojas, lo que lleva al aumento de la relación señal-ruido 3. El generador de imágenes Odyssey que usamos tiene un rango dinámico 4,5 log y una profundidad de bits de 16, Translatinga 2 16 o 65.536 tonos de infrarrojos. Esto se puede contrastar con imágenes en color de 8 bits, que sólo ofrece 2 8 o 256 tonos de color para cada longitud de onda. Por lo tanto, de formación de imágenes de 16 bits tiene una resolución más fina. Cabe señalar que las imágenes infrarrojas originales son a menudo pseudocoloreada verde (800 nm) y roja (700 nm) en los informes publicados para la presentación. Cámaras Odyssey son de uso común tanto para Western Blot e In-Cell Westerns 4-7. Dentro de la célula del Oeste en las células fijadas con formaldehído utilizan anticuerpos primarios contra cualquier proteína de interés y etiquetarlos a su vez con anticuerpos secundarios fluorescentes infrarrojos. Esta técnica es conocida por ser particularmente útil para los puntos finales de fosforilación 6. En nuestra en la celda del Oeste, que tiñe las células fijadas para las proteínas del citoesqueleto α-tubulina o microtúbulos neuronales proteína asociada 2 (MAP2) en el canal de 800 nm. Estas proteínas son lo suficientemente abundantes para producir alta relación señal-ruido. También tiñe nuestros platos en el 700canal nm para núcleos con la mancha de DRAQ5 y para el citoplasma con la mancha de zafiro. Tanto las proteínas del citoesqueleto y las DRAQ5 + manchas Sapphire por lo tanto reflejan las estructuras celulares. El tercer ensayo de viabilidad mide la función metabólica y se llama &quot;célula Titulación Glo.&quot; En este ensayo basado en la luciferasa, los valores de luminiscencia están en proporción directa a los niveles de ATP. Ensayos de ATP se utilizan comúnmente para cuantificar las células viables 8-12. Sin embargo, incluyendo la palabra &quot;título&quot; en el nombre del ensayo es algo de un nombre inapropiado porque la producción de ATP por célula puede cambiar como una función de los tratamientos de toxina y es, por tanto, no siempre en proporción al número de células 8. Los niveles de ATP también pueden verse afectados por los ritmos circadianos 13 y por la división celular y la diferenciación celular 14 15. Sin embargo, el ensayo de ATP se muestra aquí es simple de realizar y útil porque ATP es una medida robusta de metabólicaviabilidad 16-21, si no del teléfono celular número en sí. Utilizando este ensayo para complementar el infrarrojo en la celda del Oeste, por lo tanto se obtiene un panorama más completo de la integridad celular de un ensayo de cualquier solo.

<table border="0" cellpadding="0" cellspacing="0" width="821">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Cell Titer Glo</td>
			<td style="width:112px;">Promega</td>
			<td style="width:93px;">G7572</td>
			<td style="width:507px;">Buy in 100 ml quantities and aliquot, instead of purchasing the more expensive 10 ml quantity. Reconstituted, unused reagents can be refrozen at -20 &#176;C for at least 21 weeks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">18% Formalin</td>
			<td style="width:112px;">Thermo-Shandon</td>
			<td style="width:93px;">9990244</td>
			<td style="width:507px;">Buying this fixative avoids the weighing out of formaldehyde powders and boiling of the solution; exposure to vapors is thereby minimized</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sucrose</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">S0389</td>
			<td style="width:507px;">It is not essential to add this to formaldehyde solutions but it improves the appearance of the fixed cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Odyssey Block</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">927-40003</td>
			<td style="width:507px;">This fish serum can be bought in bulk and frozen at -20 &#176;C for long term use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Triton-X 100</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">21568</td>
			<td style="width:507px;">We store a stock solution of 10% Triton-X 100 in sterile water at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Monobasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S468</td>
			<td style="width:507px;">One can also buy PBS tablets or 10x PBS solutions, but they are more expensive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Dibasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S373</td>
			<td style="width:507px;">See above</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Azide (250x)</td>
			<td style="width:112px;">Ricca Chemical Company</td>
			<td style="width:93px;">7144.8-16</td>
			<td style="width:507px;">Do not buy the powder because sodium azide is very toxic. We store all our used antibodies in 1x sodium azide at 4 &#176;C until they become contaminated with debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-&#945;-tubulin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">T5168</td>
			<td style="width:507px;">This antibody is expensive but can be greatly diluted and is highly specific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-MAP2</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">M9942</td>
			<td style="width:507px;">This antibody is expensive but is highly specific (a prerequisite for In-Cell Westerns)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">800 nm Goat anti-mouse IgG</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">926-32210</td>
			<td style="width:507px;">Other companies also sell infrared secondary antibodies. Be sure to purchase the highly cross-adsorbed antibodies and note that concentrations of IgGs may vary with the source</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">DRAQ5</td>
			<td style="width:112px;">Biostatus</td>
			<td style="width:93px;">DR50200</td>
			<td style="width:507px;">This compound used to be sold by LI-COR at 1 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sapphire</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">928-40022</td>
			<td style="width:507px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Luminometer</td>
			<td style="width:129px;">PerkinElmer</td>
			<td style="width:189px;">VICTOR3 1420 multilabel counter</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Odyssey Imager</td>
			<td style="width:129px;">LI-COR</td>
			<td style="width:189px;">9201-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Shaker/Mixer</td>
			<td style="width:129px;">Research Products International</td>
			<td style="width:189px;">248555</td>
			<td></td>
		</tr>
	</tbody>
</table>

viability assays for cells in culture

Manual de células en un microscopio son un medio sensible de la evaluación de la viabilidad celular, pero consumen mucho tiempo y por lo tanto caro. Ensayos de viabilidad computarizados son costosos en términos de equipo, pero puede ser más rápido y más objetivo que el recuento de células manuales. El presente informe se describe el uso de tres de estos ensayos de viabilidad. Dos de estos ensayos son de infrarrojos y uno es luminiscente. Ambos ensayos infrarrojos se basan en un Imager 16 bits Odyssey. Un ensayo de infrarrojos utiliza la mancha de DRAQ5 para los núcleos combinados con la mancha de zafiro de citosol y se visualiza en el canal de 700 nm. El otro ensayo de infrarrojos, una en la celda Occidental, utiliza anticuerpos contra las proteínas del citoesqueleto (proteína asociada α-tubulina o microtúbulos 2) y los etiqueta en el canal 800 nm. El tercer ensayo de viabilidad es un ensayo luminiscente comúnmente utilizado para el ATP, pero usamos un cuarto del volumen recomendado para ahorrar en coste. Estas mediciones son lineales y se correlacionan con el número de CElls plateado, pero varían en sensibilidad. Todos los tres ensayos de eludir microscopía de tiempo y muestrean todo el pozo, reduciendo así el error de muestreo. Por último, todos los ensayos fácilmente se puede completar en un día del final del experimento, lo que permite un mayor número de experimentos a realizar en plazos cortos. Sin embargo, todos ellos se basan en el supuesto de que el número de células permanecen en proporción a la intensidad de la señal después de los tratamientos, la suposición de que a veces no se cumple, especialmente para ATP celular. Además, si las células aumentan o disminuyen de tamaño después del tratamiento, esto podría afectar la fuerza de la señal sin afectar el número de células. Llegamos a la conclusión de que todos los ensayos de viabilidad, incluidos los recuentos manuales, adolecen de una serie de advertencias, pero que los ensayos de viabilidad computarizados son bien vale la pena la inversión inicial. El uso de los tres ensayos en conjunto da una visión completa de la estructura y la función celular.

El factor limitante de la velocidad en estos experimentos es la tinción de infrarrojos, como el ensayo de ATP es relativamente de breve duración. Para los ensayos de infrarrojos, anticipamos que ocho placas de 96 pocillos se pueden teñir y escanean el plazo de un día a la asombrosa cifra de dos lotes de cuatro platos cada una (ver Figura 1). Esta estimación asume 20 min de la fijación, de 30 minutos de lavado, 30 min de bloqueo, 2 horas de incubación del anticuerpo primario seguido de 30 min de l...

Watch this Scientific Journal Video about Viability Assays for Cells in Culture at JoVE.com

Viability Assays for Cells in Culture

Los compuestos terapéuticos a menudo se examinaron por primera vez in vitro con los ensayos de viabilidad. Recuentos de células ciegos por un observador humano puede ser muy sensible a pequeños cambios en el número de células, pero no evaluar la función. Ensayos de viabilidad computarizados, tal como se describe aquí, pueden evaluar tanto la estructura y función de una manera objetiva.

Los ensayos de viabilidad de las células en cultivo

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Research

JoVE Journal

Biology

45.9K vistas.  Duquesne University.  Los compuestos terapéuticos a menudo se examinaron por primera vez in vitro con los ensayos de viabilidad. Recuentos de células ciegos por un observador humano puede ser muy sensible a pequeños cambios en el número de células, pero no evaluar la función. Ensayos de viabilidad computarizados, tal como se describe aquí, pueden evaluar tanto la estructura y función de una manera objetiva. 

Video: Viability Assays for Cells in Culture

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

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit ensayos para las convulsiones en la epilepsia de los mutantes Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

El análisis de Schwann-astrocito interacciones utilizando In Vitro Ensayos

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Aislamiento y cultivo primario de células hepáticas de ratas

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Evaluación de las funciones mitocondriales y la viabilidad celular renal de células que sobreexpresan la proteína quinasa C Isoenzimas

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Los ensayos acoplados para Monitoreo replegamiento de proteínas en<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente generación, por actividad humana células madre pluripotentes de células somáticas humanas con virus Sendai-

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Los ensayos para la degradación de proteínas en células de mal plegadas

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...