Ce travail a été soutenu par une subvention du NIH de recherche, une subvention de la Fondation Epilepsie et une subvention de la Fondation McKnight aux expériences sur les mouches des fruits MT drosophile ont été effectuées en conformité avec les directives et les règlements énoncés par le Comité UC Berkeley utilisation des animaux.

Préparation des échantillons: La nuit avant l&#39;expérience, les mouches mâles du génotype désiré sont anesthésiés et placés dans un flacon avec un bouchon en nylon souple. Une boule de coton serait également travailler. Après l&#39;anesthésie, le seuil épileptogène est altérée, alors les mouches sont mieux laisser pendant au moins 6 heures, bien que pendant la nuit est idéal. Le jour de l&#39;expérience, les mouches sont montées une à la fois des lames de verre avec une couche de cire dentaire sur un côté. Une seule mouche est capté par la tête à l&#39;aide d&#39;une aiguille de calibre 23 (avec le bout pointu coupé) attaché à une ligne de vide. La mouche est ensuite fixé à la cire sur la lame en poussant les pattes et les ailes dans la cire. Une fois que la mouche est immobilisé, la cire est poussé autour du cou et les yeux pour bien immobiliser la tête. Electrophysiologie: La mouche, qui est maintenant monté et prêt à aller est placé dans l&#39;électrophysiologie mis en place. L&#39;ensemble se compose d&#39;électrophysiologie de trois ensembles de micromanipulateurs, chacun avec une à deux électrodes de tungstène aiguisé attaché. La tension est fournie par un stimulateur qui est connecté à une unité d&#39;isolement attaché à l&#39;électrode de stimulation. L&#39;électrode d&#39;enregistrement est connecté à un pré-ampli qui à son tour est relié à un oscilloscope à stockage numérique. Alors qu&#39;il n&#39;est pas indispensable, il est utile d&#39;avoir un haut-parleur relié à l&#39;oscilloscope afin que les réponses peuvent être rendue audible. Une prise de terre est inséré dans l&#39;abdomen de la mouche. L&#39;électrode d&#39;enregistrement est inséré dans l&#39;un des muscles dorsaux longitudinale dans le thorax de la mouche. Les électrodes de stimulation sont insérés dans la tête. Un à côté de chaque œil. Le stimulateur doit avoir les paramètres suivants pour commencer: l&#39;intervalle d&#39;impulsion continu avec 2 secondes entre chaque impulsion. Il devrait y avoir aucun retard d&#39;impulsion. La largeur d&#39;impulsion doit être 0,2 ms. L&#39;oscilloscope doit avoir la fonctionnalité de stockage activé et les paramètres suivants: Temps / DIV doit être réglé sur 1ms/DIV. VOLTS / DIV devrait être fixé à deux 0.1V/DIV ou 50 mV / div. Le seuil de la fibre géante est déterminé par la prestation des impulsions uniques et notant la tension minimale requise pour obtenir une réponse. Cette tension est généralement autour de 2 V. Une fois le seuil de la fibre géante a été déterminé, arrêtez pulsation à la volée et de modifier les paramètres sur l&#39;oscilloscope: 0,2 s / div Les réglages sur le stimulateur doit être modifié pour: L&#39;intervalle d&#39;impulsion devrait être fixé à un seul. Le train doit être activé et réglé à 3 secondes. Les paramètres de forme d&#39;onde sont: 0,5 ms à impulsions 200Hz pendant 300 ms. La tension est changé pour le réglage désiré et un train à haute fréquence d&#39;impulsions est livré au cerveau de la mouche. Si une saisie n&#39;est pas induite, la trace de l&#39;oscilloscope affiche une ligne plate, après le train. Si les paramètres sont rapidement mis en revenir aux paramètres de départ, les impulsions uniques provoquera une réponse dans le système de fibre de géant. Si une saisie est induite, l&#39;oscilloscope montrent l&#39;activité de saisie-comme après le train. Si les paramètres sont rapidement passé aux paramètres d&#39;origine, les impulsions uniques ne parviendra pas à activer une réponse dans le système de fibre de géant. Les mouches ont une période réfractaire après un train d&#39;onde de stimulation à haute fréquence dans lequel la saisie comme l&#39;activité ne peut être induite. Si aucune saisie survient les mouches sont réfractaires pendant environ 7 minutes. Si une saisie est induite, les mouches ont une période réfractaire d&#39;environ 17 minutes. Lors de la détermination du seuil, d&#39;autres à haute fréquence des stimuli peuvent être livrés à la même mouche (après le moment approprié pour la période réfractaire) jusqu&#39;à ce que le seuil est déterminé ou jusqu&#39;à ce que la réponse de fibre géante devient trop faible pour voir. Après l&#39;expérience est terminée, la mouche est retiré de l&#39;électrophysiologie mis en place, libéré de la cire, et éliminés.

<ol>
	<li>Kuebler, D., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+of+seizure+susceptibility+in+Drosophila.">Modifications of seizure susceptibility in Drosophila.</a> J Neurophysiol. 83, 998-1009 (2000).</li><li>Kuebler, D., Zhang, H., Ren, X., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+suppression+of+seizure+susceptibility+in+Drosophila.">Genetic suppression of seizure susceptibility in Drosophila.</a> J Neurophysiol. 86, 1211-1225 (2001).</li><li>Song, J., Tanouye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seizure+suppression+by+shakB2,+a+gap+junction+mutation+in+Drosophila.">Seizure suppression by shakB2, a gap junction mutation in Drosophila.</a> J Neurophysiol. 95, 627-635 (2006).</li></ol>

Cette vidéo a démontré les étapes de base pour déterminer le seuil épileptogène d&#39;une mouche drosophile individuels. Au lieu d&#39;utiliser l&#39;oscilloscope numérique, un numériseur informatique peut également être utilisé. Cette technique est utile pour déterminer les effets d&#39;un médicament ou une modification génétique sur le seuil épileptogène d&#39;une mouche.

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		<th>Comment</th>
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<td>Digital oscilloscope</td>
<td>&nbsp;</td>
<td>Hitachi</td>
<td>VC-6523</td>
<td>&nbsp;</td>
<tr>
<td>Stimulator</td>
<td>&nbsp;</td>
<td>Cornerstone</td>
<td>S-900</td>
<td>&nbsp;</td>
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<td>Preamplifier</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>Electro 705</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulators</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>various</td>
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neurocircuit assays for seizures in epilepsy mutants of drosophila

Drosophila melanogaster est un outil utile pour étudier la saisie comme activité. Une variété de mutants dont les crises peuvent être induites soit par un choc physique ou la stimulation électrique est disponible pour l&#39;étude des divers aspects de l&#39;activité épileptique et de comportement. Toutes les mouches, y compris de type sauvage, va subir une saisie-comme activité si stimulés à une tension assez élevée. Saisie comme l&#39;activité est une réponse tout-ou-rien et chaque génotype a un seuil de crise spécifique. Le seuil de la saisie d&#39;un génotype spécifique de la mouche peut être modifiée soit par un traitement avec un médicament ou par la suppression génétique ou d&#39;amélioration. Le seuil est facilement mesuré par l&#39;électrophysiologie. Saisie-comme l&#39;activité peut être induite par stimulation électrique à haute fréquence livrés directement au cerveau et enregistrées par les muscles dorsaux longitudinaux (DLMS) dans le thorax. La DLMS sont innervés par une partie du système de fibre géante. A partir de basse tension, la stimulation à haute fréquence, et ensuite élever la tension par petits incréments, le seuil de saisie pour une seule mouche peut être mesurée.

Watch this Scientific Journal Video about Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila at JoVE.com

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

En utilisant la stimulation électrique à haute fréquence, la saisie-comme l&#39;activité peut être induite dans<em&gt; Drosophile</em&gt;. Cette activité est facilement enregistrées par le système de fibre géante.

Les dosages des Saisies en Neurocircuit Mutants épilepsie du Drosophile</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 ...

neurocircuit-assays-for-seizures-in-epilepsy-mutants-of-drosophila

Research

JoVE Journal

Biology

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

10.4K vues.  University of California, Berkeley. En utilisant la stimulation électrique à haute fréquence, la saisie-comme l'activité peut être induite dans Drosophile. Cette activité est facilement enregistrées par le système de fibre géante.

Vidéo: Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

Paradigmes pour la caractérisation pharmacologique de C. elegans mutants transmission synaptique

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

Recherche

Biologie

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Objectif transplantation chez le poisson zèbre et son application dans l&#39;analyse de mutants Eye

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Préparation d&#39;embryons pour la microscopie électronique des Drosophile Tube cardiaque embryonnaire

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

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.

Analyse de Schwann-astrocytes interactions à l&#39;aide In Vitro Tests

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

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

Protocole entraînement d&#39;endurance et de performance Tests longitudinales pour Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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.

Les analyses couplées pour la surveillance protéines repliement<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 ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Les analyses de viabilité de cellules en culture

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

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

Une méthode de perméabilisation de<em&gt; Drosophila</em&gt; Embryons pour dosages de petites molécules activité

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Génération de signalisés et Mutants dans Modèle sans marqueurs cyanobactérielles espèces

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...