Agradecemos Rev. Dr. Ralph Bohlmann y el Rev. Dr. James Voelz por sugerir la palabra &quot;Anastasis&quot; para describir la reversión de la apoptosis; Douglas R. Green para las células HeLa que expresan de forma estable el citocromo c-GFP; Charles M. Rudin y Eric E. Gardner para H446 células; Heather Cordero para la asistencia en el dibujo de la historieta en el video; Yee Hui Yeo valioso para el debate de este manuscrito. Este trabajo fue apoyado por un Sir Edward Youde Memorial Fellowship (HLT), la Beca Dr. Walter Szeto Memorial (HLT), beca Fulbright 007-2009 (HLT), Fundación de Investigación de Ciencias de la Vida de becas (HLT), NIH subvenciones NS037402 (JMH) y NS083373 (JMH), y el Comité de Becas de la Universidad de Hong Kong AoE / B-07/99 (MCF). Ho Lam Tang es una Fundación Shurl y Kay Curci Fellow de la Fundación de Investigación de Ciencias de la Vida.

The authors have nothing to disclose.

Anastasis se refiere al fenómeno por el cual las células que han de células activadas por vía de la muerte posteriormente revertir el proceso de muerte y sobrevivir. Aquí, hemos demostrado que imágenes de células vivas se puede utilizar para confirmar que las mismas células individuales, de hecho, pueden revertir proceso de muerte celular apoptótica en una etapa tardía, y luego continuar sobrevivir y reproducirse. Nuestros protocolos describen varias condiciones de tratamiento específicas del tipo de células...

La apoptosis (del griego, &quot;caer a la muerte&quot;) en general se supone que ser un proceso unidireccional que termina en suicidio celular 1-7. Alteración genética de genes pro-muerte como resultado la supervivencia de las células adicionales que de otro modo morirían en animales enteros, incluyendo las células que ya han iniciado el 8,9 vía de la apoptosis. Del mismo modo, las manipulaciones genéticas permiten que las células de mamíferos sanos que artificialmente Display &quot;Cómeme&quot; señales o que pierden la adherencia a su matriz extracelular para escapar de la muerte por la fagocitosis de células enteras o entosis, respectivamente 10,11. Sin embargo, nosotros y otros han demostrado que sin manipulación genética de células de mamíferos sanos normales y líneas celulares también pueden recuperarse de las primeras etapas de la apoptosis 12-15. El uso de herramientas para rastrear células individuales, hemos demostrado aún más la recuperación de las últimas etapas de la apoptosis 12,13, después de las células han pasado los puntos de control importantes que típicoly marcar el &quot;punto de no retorno&quot; 2-6. Estos puntos de control de la apoptosis etapa tardía incluyen liberación mitocondrial de citocromo c, la activación de caspasas, la fragmentación nuclear, y formación de cuerpos apoptóticos. Adoptamos una palabra compuesta griega &quot;Anastasis&quot;, que significa &quot;resurrección a la vida&quot;, para describir esta inversión de la apoptosis en el borde de la muerte celular 2-6. A menos que todo el proceso de recuperación de morir es observado por imágenes de células vivas, es difícil distinguir las células que han sido objeto anastasis a partir de células que nunca experimentaron eventos apoptóticos. Décadas de trabajo han puesto de manifiesto que las características morfológicas de suicidio celular por apoptosis son impulsados ​​por eventos bioquímicos y moleculares conservadas evolutivamente 16-19. Estos eventos promueven la auto-destrucción de las células para regular los procesos de desarrollo y homeostáticos en organismos unicelulares y pluricelulares mediante la eliminación de daños o dcélulas angerous 16-19. Mientras que las células apoptóticas pueden distinguirse fácilmente por manifestaciones morfológicas, bioquímicas y moleculares estandarizados de apoptosis 1,5,6,16,20, actualmente no se conoce ningún marcador específico para anastasis 12,13. Es importante destacar que las células que han sido objeto anastasis parecen ser las células sanas normales y células que sólo empiezan revertir apoptosis aparecer células que mueren como apoptosis 12,13. Por lo tanto, se necesitan nuevas herramientas para concluir con certeza que una célula sobrevive dado había experimentado previamente procesos apoptóticos activos. La apoptosis se asume generalmente como una cascada irreversible porque es un proceso de destrucción rápida y masiva. Si bien se podría tardar minutos en días de algunas células para iniciar la apoptosis, una vez que las mitocondrias han lanzado factores apoptogénicos como citocromo c al citosol 21,22, caspasas pueden ser activados en 5 minutos 23,24, seguidos por citoplasmática ycondensación nuclear dentro de 10 min 25-27, y la muerte celular poco después 25-27. Las caspasas activadas orquestan la apoptosis mediante la escisión e inactivación de componentes estructurales y funcionales clave para el propósito de demolición celular 2,28, tales como el inhibidor de la endonucleasa de DFF45 / ICAD 29,30. Las caspasas también activan factores pro-apoptóticos, tales como BID miembro de familia Bcl-2, que se transloca a la mitocondria para promover la liberación de citocromo C 31,32. Actividad caspasa también da lugar a la exposición de la superficie celular de fosfatidilserina como &quot;comerme&quot; señal para la promoción de inmersión de las células que mueren por los macrófagos o células vecinas a través de la fagocitosis 33. Además, eventos apoptóticos hacen mitocondrias disfuncionales, lo que altera la bioenergética celular y el metabolismo 34,35,36. Por lo tanto, la recuperación de dicha destrucción parece intuitivamente improbable. Contrariamente a la original esperarciones, las células pueden revertir el proceso de muerte celular apoptótica incluso en una etapa tardía. Al monitorear continuamente el destino de morir las células en cultivo, se observó la reversibilidad de la apoptosis etapa tardía en una gama de células primarias y líneas celulares 12,13. La eliminación del estímulo muerte permitió la recuperación de las características evidentes de la apoptosis, tales como la fragmentación mitocondrial, condensación de la cromatina, el daño del ADN, formación de ampollas en la membrana plasmática, la exposición de la superficie celular de la fosfatidilserina, la liberación de citocromo c mitocondrial, la activación de caspasas, la fragmentación nuclear, contracción celular, y formación de cuerpos apoptóticos. Estas observaciones plantean preguntas sin respuesta sobre las funciones, las consecuencias y los mecanismos de anastasis. Para abordar estas cuestiones, es un requisito previo para identificar de forma fiable las células que han sido objeto anastasis. A continuación, describimos los métodos de microscopía en vivo y un biosensor para la detección de caspasa células que han invertido previamente apoptosis etapa tardía y XXen sobrevivió.

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strategies for tracking anastasis, a cell survival phenomenon that reverses apoptosis

Anastasis (del griego, &quot;el aumento de la vida&quot;) se refiere a la recuperación de las células que mueren. Antes de que estas células se recuperan, que han pasado a través de los puntos de control importantes de la apoptosis, incluyendo la fragmentación mitocondrial, la liberación de citocromo c mitocondrial en el citosol, la activación de las caspasas, condensación de la cromatina, el daño del ADN, fragmentación nuclear, formación de ampollas en la membrana plasmática, el encogimiento celular, la exposición de la superficie celular de fosfatidilserina, y la formación de cuerpos apoptóticos. Anastasis puede ocurrir cuando los estímulos apoptóticos se eliminan antes de la muerte, permitiendo así que las células que mueren para revertir la apoptosis y potencialmente otros mecanismos de muerte. Por lo tanto, anastasis parece implicar procesos de curación fisiológicos que también podrían sostener las células dañadas de manera inapropiada. Las funciones y mecanismos de anastasis aún no están claros, obstaculizada en parte por las limitadas herramientas para la detección de eventos pasados ​​después de la recuperación de las células aparentemente sanas. Estrategias para detectar anastasis permitirá a los estudios de los mecanismos fisiológicos, los peligros de las células no-muertos en patología de la enfermedad y posibles terapias para modular anastasis. A continuación, describimos las estrategias eficaces usando microscopia de células vivas y un biosensor caspasa mamífero para identificar y rastrear anastasis en células de mamíferos.

Para estudiar la reversión de la apoptosis, las células de cultivo de tejidos se expusieron primero a un estímulo muerte para activar la apoptosis. Cuando las células exhiben características de apoptosis, medio de cultivo fresco se aplica entonces para lavar el estímulo y luego se incuban las células que mueren para permitir la recuperación (Figura 1A). Aquí, la cuestión clave está abordando es hasta qué punto las células cultivadas que mueren individuales pueden progresar hacia la apoptosi...

Watch this Scientific Journal Video about Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis at JoVE.com

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Estrategias para Anastasis Seguimiento, una célula de supervivencia Fenómeno que Invierte Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

strategies-for-tracking-anastasis-cell-survival-phenomenon-that

Research

JoVE Journal

Biology

17.9K vistas.  Johns Hopkins University Bloomberg School of Public Health. The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis. 

Video: Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Seguimiento de los cambios dinámicos en niveles de calcio mitocondrial de la apoptosis mediante un sensor de calcio genéticamente codificados

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Investigación

Biología

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

Modificado anexina V / yoduro de propidio ensayo Apoptosis una evaluación precisa de la muerte celular

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

La activación de la apoptosis mediante microinyección citoplasmática del citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

El seguimiento de deformaciones morfogenética del tejido en el embrión de pollo temprano

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Las formulaciones para la liofilización de las bacterias y su influencia en la supervivencia celular

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimizado manchas y Proliferación métodos de modelización para la vigilancia del uso de tintes División Celular Celulares de seguimiento

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Celular seguimiento mediante fotoconvertible proteínas durante el desarrollo de pez cebra

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

El tomate / GFP-FLP / FRT Método para imágenes en vivo del mosaico adultos<em&gt; Drosophila</em&gt; Las células fotorreceptoras

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

Una guía para la moderna cuantitativa fluorescente Western Blot con las estrategias de solución de problemas

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Evaluación del selectivo ARNm traducción en células de mamífero por polisoma perfiles

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...