KO wird durch einen Zuschuss von der American Heart Association unterstützt.

Bevor Sie beginnen Adult Herzen <ol><li> Frisch vorzubereiten künstliche Hämolymphe (AH) Lösung mit 108mm Na +, 5 mM K +, 2 mM Ca 2 +, 8mm MgCl 2, 1 mM NaH 2 PO 4, 4mm NaHCO 3, 10 mM Saccharose, 5 mM Trehalose, 5 mM HEPES (pH 7,1, alle Reagenzien von Sigma Chemicals). Die Saccharose und Trehalose sollte auf die AH aus dem Kühlschrank Stammlösungen hinzugefügt gerade vor Gebrauch, um bakterielle Verunreinigungen zu verhindern. </li><li> Bringen AH auf Raumtemperatur und mit Sauerstoff die Lösung durch Luft-Blasen für mindestens 15 min. </li><li> Ziehen Sie mehrere feine Kapillaren (z. B. Glaskapillaren, 100 ul, VWR) zur Entfernung von Fett benötigt. </li></ol> Larven Herzen <ol><li> Sylgard-beschichtete Deckgläser: mit einem Zahnstocher, Transfer über 50-70μl der Sylgard Lösung auf eine 22x22mm Deckglas. Lassen Sie die Sylgard härten bei 65 ° C über Nacht. </li><li> Bereiten Sie die Broadie und Bate-Puffer (135mm NaCl, 5 mM KCl, 4mm MgCl 2, 2 mM CaCl 2, 5 mM TES, 36mm Sucrose;. PH 7,15 Siehe Broadie und Bate, 1993). </li><li> Bereiten Sie die notwendigen Tools, um die Histoacryl Klebstoff anwenden. Ziehen Sie zunächst mehrere feine Kapillaren (zB Science Products GB100T8P). Schließen Sie ein 1,5 ml Pipettenspitze mit einem kleinen Plastikschlauch (Tygon, 1 / 32 &quot;), indem Sie das kleinere Ende in die Röhre. Legen Sie eine Kapillare mit der weiten Öffnung am anderen Ende. Laden Sie die Kapillare mit Histoacryl Kleber durch Eintauchen der Kapillare in einem Tropfen Klebstoff und sanfte Saugwirkung in die Spitze aus Kunststoff. Bei der Arbeit mit dem Klebstoff sollte man eine zweite Glaskapillare zur Hand haben, um übermäßige Kleber von der Spitze des Klebers Kapillare zu entfernen. Der Kleber ist flüssig, bis es in Kontakt mit Kochsalzlösung, wo es härtet innerhalb von ein paar Sekunden. Aber es bleibt in der Regel aus Kunststoff lange genug, um aus dem Leim Kapillarspitze oder während ihrer Anwendung auf die Probe entfernt werden. </li></ol> Semi-intakt Drosophila Herz aus dem Adult Fly <ol><li> Adult Drosophila-Fliegen werden anästhesiert mit Fly Nap (Carolina Biological Supply Co.) für 2-5 Minuten. Vorsicht ist geboten, um die Fliegen in der Fly Nap für längere Belichtungszeiten zu verlassen. Kurzfristige Exposition gegenüber Kälte kann auch verwendet werden, um Fliegen zu betäuben, aber CO 2 wird nicht empfohlen, da es länger hat nachhaltige Auswirkungen auf die Herzfrequenz und Rhythmik. </li><li> Narkotisierten fliegt platziert sind, dorsalen Seite nach unten in eine Petrischale mit einer dünnen Schicht Vaseline beschichtet. Die hydrophoben Cuticula in den Flügeln und Körper reversibel &quot;kleben&quot; an das Gelee, aber die fliegen können neu positioniert, wenn nötig. Dies wird besonders wichtig bei der späteren Manipulationen. </li><li> Eine erste Schnitt erfolgt mit einer gebogenen Feder Schere (Roboz # 5611). Die Scherenblätter sind unter die Beine gelegt und nach unten in Richtung der dorsalen Fläche des Brustkorbes in der Nähe des Halses abgewinkelt. Der Kopf, Bauchmark und Beine sind mit einem einzigen Schnitt entfernt. Das Präparat wird dann in ein mit Sauerstoff angereichertes, künstliche Hämolymphe (AH)-Lösung getaucht. </li><li> Mit Feder Schere der hinteren Spitze des Bauches (bestehend aus Abdominalsegmente 7 und 8) ist mit einem einzigen Schnitt entfernt. Dies ermöglicht den Zugang zur Herstellung von seitlichen Einschnitte an den beiden Rändern des Bauches und dient dazu, die Verbindung zwischen dem hinteren Bauch und der Bauch Nagelhaut zu durchtrennen. Die befreiten Klappe ventral abdominal Nagelhaut wird dann entfernt. </li><li> In den Schritten 3 und 4 des vorderen und hinteren Anschlüsse des Darms werden durchtrennt, so dass die Bauchorgane sind jetzt an Stelle nur durch tracheols statt. Der Darm und andere Bauchorgane können in der Regel als eine einzige Masse mit Juwelier Zange (Roboz, # 55) und sanft zerren entfernt werden. Das Entfernen der inneren Organe zeigt das schlagende Herz Rohr noch an der dorsalen Kutikula durch Fettkörper umgeben befestigt. </li><li> Die Fettkörper sind ziemlich undurchsichtig im Lichtmikroskop folglich ist es oft notwendig, um etwas von diesem Fett zu entfernen, um das Herz Rohr deutlich zu sehen. Dies wird durch eine Fettabsaugung Technik mit fein gezeichneten Glaskapillaren durchgeführt. Kapillaren werden mit einer Standard-Pipette Abzieher (zB Sutter P-97 Pipette Puller) und werden dann in Kunststoff-Schlauch (Tygon, 1 / 16 &quot;) befestigt, um ein Vakuum Quelle eingefügt. Dieses System ist zur Absaugung von überschüssigem Fett um das Herz Rohr eingesetzt. Saugkraft ist proportional zur Spitze Durchmesser so Pipetten mit Spitzen von mehr als 40 Mikrometer im Durchmesser nicht verwendet werden sollte. Extreme Vorsicht ist zu vermeiden, berühren das Herz selbst und werden, weil der hintere Teil des Herzens ist sehr zerbrechlich dieser Region insgesamt sollte vermieden werden. </li><li> Die erwachsenen Drosophila Herzen Röhre ist nun frei und sollte zu schlagen. Bei Bedarf kann die Nagelhaut und befestigt Herz kann durch leichtes Anheben der Oberhaut aus der Vaseline und sorgfältig Stopf es wieder nach unten, wieder jeglichen Kontakt mit dem Herzgewebe neu positioniert werden. Bei tseine Stelle die Lösung Baden der Zubereitung sollte mit frischem AHL ersetzt werden. Dieses Präparat sollte gestattet werden, für 20-30 Minuten mit Sauerstoffanreicherung vor Manipulationen Gleichgewicht sein. Wenn die Zubereitung nicht länger als 60 Minuten gehalten werden, sollte es mit frischen AHL perfundiert werden. </li></ol> Immobilisierung Drosophila-Larven für die optische Aufzeichnung <ol><li> Geben Sie einen Tropfen Histoacryl Kleber in der Nähe von jeder Ecke des Sylgard Deckgläser. Add 50 ul B &amp; B-Puffer auf den Leim Tropfen, die sich verhärten sofort. Füllen Sie den Raum zwischen den geklebten Stellen mit B &amp; B - der Leim der Puffer auf der Folie halten. </li><li> Nehmen Sie eine Wanderung L3 Larve und legen Sie es auf ein Stück nasse Seidenpapier auf Eis für 2 min. Dies verlangsamt die Bewegung. Übertragen Sie anschließend die Larve in den Puffer und richten Sie ihn mit dem Rücken nach oben (dies ist die Seite der Larve, die denticle Gürtel fehlt). Schnell mit Leim zwischen dem Kopfbereich und dem Sylgard. Der Kleber härtet sehr schnell und verhindern, dass der Kopf der Larve von bewegten. </li><li> Mit einer Pinzette vorsichtig packen das Tier von der hinteren Luftlöcher und sanft dehnen. Fix die Larve in dieser Position durch die Anwendung des Klebers auf beiden Seiten der Larve an der hinteren Hälfte. Um eine weitere Bewegung der Kutikula durch die Wand des Körpers Muskeln zu verringern, fügen Sie mehr Leim an den Flanken des Tieres. Die Larve ist nun in dieser Position fixiert. Das Deckglas kann in eine kleine Petrischale mit AHL oder PBS gefüllt platziert werden. Herzschläge können nun ohne Einmischung von Körper Wand Bewegungen aufgezeichnet werden. </li></ol> Repräsentative Ergebnisse: Die semi-intakten, erwachsenen Drosophila Herz rhythmisch schlagen für Stunden nach Dissektion, wenn in frischen, sauerstoffreichen AH gepflegt (siehe Ergänzende Daten, Ocorr, et al., 2007). Ein repräsentatives Beispiel ist in Movie 1 dargestellt. Herz vom 3. Larvenstadium Larve und jungen Fliegen (1 bis 3 Wochen nach Schlüpfen) weisen in der Regel eine regelmäßige Herzschlag mit Raten zwischen 1 bis 3 Hz. Flies älter als 3 Wochen in der Regel mehr Arrhythmie, dennoch sind diese Herzen noch in der Lage, spontan zu schlagen über Stunden in Sauerstoff AHL. Hearts in erwachsenen Fliegen, die als Folge der Dissektion Verfahren beschädigt sind in der Regel zeigen lokalisierten Regionen extreme Verengung, die nicht in der Lage zu entspannen. Damit solche Präparate gelten als beschädigt werden und werden verworfen; Heartbeat Preise langsamer als 1 Hz sind nur selten in Fliegen jünger als 3 Wochen alt beobachtet. <a href="http://www.jove.com/files/ftp_upload/1425/MOVIE1.avi">Klicken Sie hier, um Movie 1 herunterladen.</a> - A eine Woche alt erwachsenen Wildtyp fly (w 1118 Labor-Stamm), die den exponierten Bauch-Herz Rohr (anterior nach links). Beachten Sie regelmäßige, rhythmische Kontraktionen. Opaque Regionen in der oberen rechten Ecke werden die restlichen Bauchfett Stellen; runde Zellen auf beiden Seiten des Herzens sind pericardial Zellen.

<ol>
	<li>Ocorr, K., Reeves, N., Wessells, R. J., Fink, M., Chen, H. -. S. V., Akasaka, T., Yasuda, S., Metzger, J., Giles, W., Posakony, J. W., Bodmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNQ+potassium+channel+mutations+cause+cardiac+arrhythmias+in+Drosophila+that+mimic+the+effects+of+aging.">KCNQ potassium channel mutations cause cardiac arrhythmias in Drosophila that mimic the effects of aging.</a> Proc Natl Acad Sci U S A. 104, 3943-3948 (2007).</li><li>Broadie, K. S., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+embryonic+neuromuscular+synapse+of+Drosophila+melanogaster.">Development of the embryonic neuromuscular synapse of Drosophila melanogaster.</a> J Neurosci. 13, 144-166 (1993).</li></ol>

Die Drosophila-Modell hat sich als ein leistungsfähiges Tool, das genetische verwendet wurde, um eine Vielzahl von wissenschaftlichen Fragestellungen reichen von embryonalen Entwicklung, Lernen und Gedächtnis-Adresse sein. Kürzlich dieses vielseitige Modell-Organismus wurde verwendet, um die Genetik der Herzfunktion in-the-fly zu untersuchen. Eine Reihe von Versuchen, Herz Physiologie bei erwachsenen Drosophila zu quantifizieren sind auf Beobachtungen in intakten fliegt durch die Bauchdecke Nagelhaut, gelten. Die mei...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Micro Dissecting Spring Scissors (curved)</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5611</td>
<td>Good for gross cuts (Step 3)</td>
</tr>
<tr>
<td>Micro Dissecting Spring Scissors (straight)</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5620</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #55 forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11295-51</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Glass Capillaries, 100ul</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53432-921</td>
<td>&nbsp;</td>
<tr>
<td>Glass Capillaries, fine</td>
<td>&nbsp;</td>
<td>Science Products</td>
<td>GB100T8P</td>
<td>&nbsp;</td>
<tr>
<td>Pipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Equipment</td>
<td>P-97</td>
<td>Both horizontal and vertical pipette pullers will work</td>
</tr>
<tr>
<td>Plastic tubing</td>
<td>&nbsp;</td>
<td>Tygon R-3603</td>
<td>1/16&#x201D; inner diameter</td>
<td>for 100&mu;l capillaries</td>
</tr>
<tr>
<td>Plastic tubing</td>
<td>&nbsp;</td>
<td>Tygon R-3603</td>
<td>1/32&#x201D; inner diameter</td>
<td>for small capillaries</td>
</tr>
<tr>
<td>Histoacryl&reg; tissue adhesive</td>
<td>&nbsp;</td>
<td>B.Braun, Melsungen</td>
<td>&nbsp;</td>
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visualizing the beating heart in drosophila

Die<em&gt; Drosophila</em&gt; Herz hat vor kurzem als ein gutes Modellsystem für die Untersuchung der genetischen, zellulären und molekularen Mechanismen, die Funktion in myogene Herzen entstanden. Ein wichtiger Schritt bei der Untersuchung der Herzfunktion in-the-fly ist, einen Weg in das Herz in einer Weise, dass seine myogene Funktion bewahrt, solange noch die schlagenden Herzen Organ beobachtet und protokolliert werden zugreifen. Zwei verschiedene Methoden zur Beobachtung und Aufzeichnung des schlagenden Herzens in beiden Larven und erwachsenen<em&gt; Drosophila</em&gt; Werden hier beschrieben. Unsere semi-intakten Präparat mit adulten Fliegen ermöglicht die übersichtliche Visualisierung der abdominalen Herz ohne Störung durch die pigmentierte Haut-und darüberliegenden Fettkörper. Zur Aufnahme Larven Herz schlägt, ist es notwendig, die Larve, die Körperwand Bewegungen wodurch Herz Bewegungen, die nicht mit myokardialen Kontraktionen verbunden sind minimiert immobilisieren. Unsere Methoden, stabile Erwachsene und Larven Herzen Zubereitungen, die stundenlang mit Raten von 1-3 Hz schlagen können.

Watch this Scientific Journal Video about Visualizing the Beating Heart in Drosophila at JoVE.com

Visualizing the Beating Heart in Drosophila

Technik für die Visualisierung des schlagenden Herzens in Larven und erwachsene erforderlich<em&gt; Drosophila</em&gt; Präsentiert werden. Jede Lebensphase erfordert eine andere Methodik.

Visualisierung des schlagenden Herzens in Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

visualizing-the-beating-heart-in-drosophila

Research

JoVE Journal

Biology

Visualizing the Beating Heart in Drosophila

18.6K Aufrufe.  The Sanford Burnham Institute for Medical Research. Technik für die Visualisierung des schlagenden Herzens in Larven und erwachsene erforderlich Drosophila Präsentiert werden. Jede Lebensphase erfordert eine andere Methodik.

Serie: Visualizing the Beating Heart in Drosophila

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualisierung der Live- Drosophila Glial-neuromuskulären Synapse mit Fluoreszenzfarbstoffen

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Forschung

Biologie

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Fluoreszenzmarkierung Drosophila Herz Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Überwachung Herzfunktion Larven Drosophila melanogaster Für physiologische Untersuchungen

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

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 ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Die Quantifizierung der γH2AX Foci in Response to ionisierender Strahlung

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

Die Quantifizierung der γH2AX Foci in Gewebeproben

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

Auswertung der räumlichen Verteilung der γH2AX folgenden ionisierender Strahlung

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Klonogene Test: adhärenter Zellen

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

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  ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Bewertung von Calcium in Sparks Intact Skelettmuskelfasern

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...