Wir bedanken uns für die Unterstützung zu BHR durch ein Discovery-Stipendium sowie ein Recherche-Tools und Instrumenten-Stipendium der Natural Sciences and Engineering Research Council of Canada (NSERC). Wir erkennen auch H. Oda und die Bloomington Drosophila Lager-Center für die Bereitstellung von genetischen Aktien, die im Beispiel Live-Imaging-Sequenzen verwendet wurden.

Vorbereitung <ol><li> Sammeln Embryonen auf Standard Traubensaft Agar Platten 4. Es ist zweckmäßig, eine automatisierte Drosophila Egg Collector (flymax Scientific Equipment Ltd) zu verwenden. Verwenden von synchronen inszeniert Embryo Sammlungen wird die Zahl der dechorionations, die durchgeführt werden, um eine ausreichende Anzahl von Embryonen in der gewünschten Entwicklungsstadium zu erhalten muss zu reduzieren. </li><li> Bereiten Sie eine dechorionation gleiten durch die Anbringung eines Stück doppelseitigem Klebeband auf einen Objektträger, ziehen Sie die Schutzfolie vom Band. </li><li> Bereiten Sie die Live-Imaging-Kammer, indem ein Stück des Gewebes in der auch der Kammer passen. Legen Sie das Gewebe in den Brunnen und nass ist es mit destilliertem Wasser. </li><li> Mix Halocarbonöl (Halocarbon Products Corp) Viskosität Serie 700 und Serie 56 mit einem Verhältnis von 1:1. Die Mischung kann gespeichert und verwendet werden auf unbestimmte Zeit. </li><li> Legen Sie mehrere kleine Tropfen Halocarbonöl auf der Oberfläche von einem Deckglas (22 X 40 mm, Nr. 2). Die Lautstärke ist nicht kritisch, sollte aber in der Reihenfolge von 20-40 ul werden. </li></ol> Verfahren <ol><li> Mit Hilfe von einem Stereomikroskop, sammeln Embryonen aus der Traubensaft-Agar-Platten entweder mit einem Paar von Juwelier Zange (Nr. 5) oder eine sehr feine Pinsel. Embryonen neigen dazu, miteinander und mit der Zange &quot;Spitzen-Stick, sie sind leicht in Klumpen zusammen. </li><li> Mit Hilfe von einem Stereomikroskop, sanft niedriger die Klumpen von Embryonen, die zu den Spitzen der Pinzette auf die Oberfläche des Bandes auf der dechorionation schieben müssen eingehalten werden. </li><li> Wieder mit dem Stereomikroskop, sanft anstoßen oder Schlaganfall die Embryonen mit der Seite der Zange um Aufbrechen der äußeren wachsartige Chorion ohne Zerreißen der inneren Dotterhaut (der Embryo wird platzen, wenn die Dotterhaut ist gebrochen). </li><li> Sobald die äußere Chorion wurde gebrochen hat, necken sich der Embryo aus dem Chorion, der dechorionated Embryo neigt dazu, an die Oberfläche der Zange zu halten. Achten Sie darauf, berühren Sie nicht die dechorionated Embryo auf der Oberfläche des Bandes. </li><li> Sobald ein Embryo dechorionated ist, schnell zu übertragen, um die zuvor hergestellte Tropfen Halocarbonöl auf dem Deckglas. Berühren Sie die Spitze der Zange Durchführung der dechorionated Embryo in die Oberfläche des Halocarbonöl drop - das löst den Embryo aus der Zange in das Öl. </li><li> Bestätigen Übertragung des Embryos auf die Öltropfen. Dies kann mit dem bloßen Auge, sofern Sie über einen schwarzen Hintergrund arbeiten und eine faseroptische Lichtquelle in einem schrägen Winkel gesetzt getan werden. </li><li> Bevor Sie versuchen, ein anderer Embryo dechorionate, sauber kein Öl aus der Zange. Pinzetten in Öl überzogen haben weniger Kauf auf dem Chorion Oberfläche, so dass dechorionation erschwert. </li><li> Dechorionated Embryonen sind lebhaft in der Halocarbonöl. Mit einer Pinzette oder einem Pinsel und während Sie durch ein Stereomikroskop, drücken Sie den Embryo an der Unterseite der Öltropfen und ordnen sie in die gewünschte Position. </li><li> Schnell umdrehen das Deckglas über dem Brunnen der Live-Imaging-Kammer. Bestätigen Sie, dass die Embryonen vor dem Deckglas ruht in der gewünschten Orientierung. Durch den Auftrieb im Öl, wird die Embryonen nun gegen die untere Fläche des Deckglases zu schweben. Befestigen Sie das Deckglas auf die Live-Imaging-Kammer mit Klebeband. Lüften Sie den Raum, indem Löcher in das Band in der Gegend, wo das Band erstreckt sich über die Lücke zwischen dem Ende des Deckglases und das Ende der gut von der Live-Imaging-Kammer. </li><li> Eine aufrechte Fluoreszenzmikroskop oder einem Fluoreszenz-Stereomikroskop kann nun verwendet werden, um Bild die Embryonen werden. </li></ol>

<ol>
	<li>Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L., Montague, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forces+contribute+to+cell+sheet+morphogenesis+for+dorsal+closure+in+Drosophila.">Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila.</a> J. Cell Biol. 149, 471-490 (2000).</li><li>Schock, F., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+processes+associated+with+germ+band+retraction+in.">Cellular processes associated with germ band retraction in.</a> , 248-2429 (2002).</li><li>Reed, B. H., Wilk, R., Schock, F., Lipshitz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-dependent+apposition+of+Drosophila+extraembryonic+membranes+promotes+morphogenesis+and+prevents+anoikis.">Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis.</a> Curr. Biol. 14, 372-380 (2004).</li><li>Wieschaus, E. p., Nusslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: a practical approach. , 199-227 (1986).</li></ol>

Wir beschreiben eine neue Methode zur Herstellung von Drosophila-Embryonen für die Live-Imaging-Analyse, die wir als die hängenden Tropfen Protokoll. Leider ist es nicht möglich, den hängenden Tropfen-Protokoll verwenden, wenn die Arbeit mit einem inversen Mikroskop. In diesem Fall wird die Sandwich-Technik (wie in der abstrakten oben beschrieben) verwendet werden muss und Kompression der Embryonen weiterhin Anlass zur Sorge. In Experimenten, bei denen Zellen Form und Größe gemessen, u...

Standard-Ausrüstung und Materialien benötigt, um Embryonen für Live-Imaging-Vorbereitung mit dem hängenden Tropfen Protokoll umfassen die folgenden Punkte: Objektträger, Deckgläser (22 x 40 mm, Nr. 2), doppelseitiges Klebeband, Juwelier Pinzetten (Dumont Stil Nr. 5 ), Halocarbonöl Serie 56 und Serie 700 (Halocarbon Products Corp), einem Messer oder einzelne Rasierklinge, Seidenpapier, Schere, destilliertes Wasser, Allzweck-Klebeband und einer Pipette geeignet zur Förderung von 20-40 ul. Die Live-Imaging-Protokoll erfordert auch eine maßgeschneiderte Live-Imaging-Kammer. Dies wird durch das Schneiden eines 5 mm denke Polycarbonat Kunststoffplatten an die Dimensionen eines Standard-Objektträger (75 x 25 mm) und mit einem Rotor zu einem 3mm tiefe Depression in der Folie (20 X 55 mm) erstellen vorbereitet. Der Zugang zu einem Stereomikroskop (Binokular) und eine faseroptische Lichtquelle ist ebenfalls erforderlich.

the preparation of drosophila embryos for live-imaging using the hanging drop protocol

Das grün fluoreszierende Protein (GFP)-basierte Zeitraffer Live-imaging ist eine leistungsstarke Technik für die Untersuchung der genetischen Regulation von dynamischen Prozessen wie Gewebemorphogenese, Zell-Zell-Adhäsion oder Zelltod. Drosophila-Embryos, die GFP sind leicht abgebildet entweder mit stereoskopischen oder konfokalen Mikroskopie. Ein Ziel eines jeden Live-imaging-Protokoll ist es, schädliche Auswirkungen wie Dehydratation und Hypoxie zu minimieren. Vorherige Protokolle für die Vorbereitung Drosophila-Embryonen für die Live-imaging-Analyse beteiligt Platzierung dechorionated Embryonen in Halocarbonöl und sandwiching sie zwischen einem Halogenkohlenwasserstoff gasdurchlässige Membran und einem Deckglas<sup&gt; 1-3</sup&gt;. Die Einführung der Kompression durch die Montage Embryonen auf diese Weise stellt eine unerwünschte Komplikation für biomechanische-basierte Analyse der Morphogenese. Unsere Methode, die wir als die hängenden Tropfen Protokoll, ergeben sich ausgezeichnete Lebensfähigkeit von Embryonen während der Live-Bildgebung und verlangt nicht, dass Embryonen komprimiert werden. Kurz gesagt, beinhaltet die hängenden Tropfen Protokoll der Platzierung von Embryonen in einem Tropfen Halocarbonöl, die aus einem Deckglas, das ist wiederum in der Position über eine feuchte Kammer fest aufgehängt ist. Neben der Bereitstellung von Gasaustausch und verhindert Austrocknung, nimmt diese Anordnung den Vorteil des Auftriebs von Embryonen in Halocarbonöl sie davon abzuhalten, aus der Position im Zeitraffer Übernahme zu verhindern. Dieses Video beschreibt detailliert, wie zu sammeln und vorzubereiten Drosophila-Embryonen für die Live-Aufnahme nach dem hängenden Tropfen Protokoll. Dieses Protokoll eignet sich für die Bildgebung dechorionated Embryonen mit Stereomikroskopie oder aufrechte Verbindung Fluoreszenzmikroskop.

Watch this Scientific Journal Video about The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol at JoVE.com

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Eine einfache, kostengünstige und effektive Methode zur Herstellung von Drosophila-Embryonen für die Live-imaging-Analyse vorgestellt. Unser Protokoll sieht vor Feuchtigkeit und Gasaustausch und komprimiert nicht die Drosophila-Embryo. Diese Methode eignet sich für GFP-basierte Live-Darstellung von Drosophila-Embryonen mit einem Stereomikroskop oder aufrecht zusammengesetzte Mikroskop.

Die Herstellung Drosophila Embryonen für Live-Imaging mit dem Hanging-Drop-Protokoll

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

the-preparation-drosophila-embryos-for-live-imaging-using-hanging

Research

JoVE Journal

Biology

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

15.0K Aufrufe.  University of Waterloo. Eine einfache, kostengünstige und effektive Methode zur Herstellung von Drosophila-Embryonen für die Live-imaging-Analyse vorgestellt. Unser Protokoll sieht vor Feuchtigkeit und Gasaustausch und komprimiert nicht die Drosophila-Embryo. Diese Methode eignet sich für GFP-basierte Live-Darstellung von Drosophila-Embryonen mit einem Stereomikroskop oder aufrecht zusammengesetzte Mikroskop.

Serie: The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Vorbereitung der neuronalen Kulturen aus Midgastrula Bühne Drosophila Embryonen

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Forschung

Biologie

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

In-vitro-Differenzierung von embryonalen Stammzellen der Maus (mES) Zellen unter Verwendung des Hanging-Drop-Methode

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Live-Imaging von Gliazellen Migration in der Drosophila Eye Imaginalscheibe

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Live-Imaging der Zebrafisch embryonalen Gehirn durch konfokale Mikroskopie

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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.

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

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

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Live-Bildgebung Drosophila melanogaster Embryonale Hemocyte Migrationen

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Erstellung von Entwicklungs-und Adult Drosophila Brains und retinae für Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Der Ausarbeitung der einzelnen Drosophila Ei Chambers für Live-Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

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.

Live-Imaging von apoptotischen Ausverkauf während<em&gt; Drosophila</em&gt; Embryogenese

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

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.

Die Tomate / GFP-FLP / FRT Verfahren zur Live-Imaging von Mosaic Erwachsene<em&gt; Drosophila</em&gt; Sehzellen

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