KO è sostenuto da un finanziamento della American Heart Association.

Prima di iniziare Cuori adulti <ol><li> Appena preparare emolinfa artificiale (AH) soluzione contenente 108mm Na +, 5mm K +, 2mm Ca 2 +, 8 mm MgCl 2, 1 mM NaH 2 PO 4, 4mm NaHCO 3, saccarosio 10mM, trealosio 5mm, HEPES 5mm (pH 7.1, tutti i reagenti da Prodotti chimici Sigma). Il saccarosio e trealosio dovrebbero essere aggiunti alla AH dalle soluzioni magazzino refrigerato al momento dell&#39;uso, al fine di prevenire la contaminazione batterica. </li><li> AH portare a temperatura ambiente e ossigenare la soluzione da bolle d&#39;aria per almeno 15 min. </li><li> Tirare diversi fini capillari (ad esempio vetro Capillari, 100ul, VWR) necessari per la rimozione di grasso. </li></ol> Cuori larvale <ol><li> Sylgard rivestita coprioggetto: usando una, trasferimento stuzzicadenti circa 50-70μl di soluzione Sylgard su un vetrino 22x22mm. Lasciate che i indurire Sylgard a 65 ° C durante la notte. </li><li> Preparare il Broadie e tampone del Bate (135mm NaCl, KCl 5mm, 4mm MgCl 2, 2mM CaCl 2, TES 5mm, Saccarosio 36mm;. PH 7,15 Vedi Broadie e Bate, 1993). </li><li> Preparare gli strumenti necessari per applicare la colla histoacryl. In primo luogo, tirare diversi fini capillari (ad esempio Science Prodotti GB100T8P). Collegare la punta della pipetta 1,5 ml con un tubo di plastica di piccole dimensioni (Tygon, 1 / 32 &quot;), inserendo l&#39;estremità più piccola nella provetta. Inserire un capillare con la sua ampia apertura verso l&#39;altra estremità. Caricare il capillare con colla Histoacryl immergendo il capillare in una goccia di colla e di aspirazione delicata nella punta di plastica. Quando si lavora con la colla si dovrebbe avere un secondo bicchiere capillare a portata di mano per rimuovere la colla eccessiva dalla punta della colla capillare. La colla è liquida fino a quando non entra in contatto con soluzione fisiologica tamponata dove si indurisce entro un paio di secondi. Tuttavia, rimane di solito in plastica abbastanza a lungo per essere rimossi dalla punta capillare colla o durante l&#39;applicazione al modello. </li></ol> Semi-intatta Cuore Drosophila dal Fly adulti <ol><li> Adulto vola Drosophila sono anestetizzati con Nap Fly (Carolina Biological Supply Co.) per 2-5 minuti. Bisogna fare attenzione a non lasciare le mosche nel Nap Fly per esposizioni più lunghe. Esposizione a breve termine al freddo può anche essere utilizzato per anestetizzare le mosche, ma di CO 2 non è raccomandato in quanto ha più effetti duraturi sulla frequenza cardiaca e ritmicità. </li><li> Vola anestetizzati sono posti, lato dorsale verso il basso, in una piastra di Petri rivestiti con un sottile strato di vaselina. La cuticola idrofobo nelle ali e il corpo reversibile &quot;bastone&quot; per la gelatina, ma il volo può essere riposizionato, se necessario. Questo diventa particolarmente importante durante le manipolazioni successive. </li><li> Un primo taglio è realizzato con un paio di forbici curve primavera (Roboz # 5611). Le lame a forbice sono posti sotto le gambe e angolati verso il basso verso la superficie dorsale del torace, vicino al collo. La testa, cordone nervoso ventrale, e le gambe vengono rimossi con un unico taglio. La preparazione viene poi immerso in un ossigenato, emolinfa artificiale (AH) soluzione. </li><li> Utilizzando le forbici molla la punta posteriore dell&#39;addome (formato da segmenti addominali 7 e 8) viene rimosso con un unico taglio. Questo consente l&#39;accesso di questi tagli, lungo entrambi i bordi laterali dell&#39;addome e serve a recidere il legame tra l&#39;intestino posteriore e la cuticola addominale. Il lembo libero della cuticola ventrale addominale viene quindi rimosso. </li><li> Nei passaggi 3 e 4 le connessioni anteriore e posteriore dell&#39;intestino sono così reciso gli organi addominali sono ora tenuti in posizione da solo tracheols. L&#39;intestino e altri organi addominali di solito può essere rimosso come una massa unica con pinze gioielleria (Roboz, # 55) e gentile strattoni. La rimozione degli organi interni rivela il tubo cuore pulsante ancora attaccato alla cuticola dorsale circondato da corpi grassi. </li><li> I corpi grassi sono abbastanza opachi al microscopio ottico, di conseguenza è spesso necessario per rimuovere una parte di questo grasso per vedere il tubo di cuore in modo chiaro. Questo si ottiene utilizzando una tecnica di liposuzione finemente disegnate vetro capillari. Capillari sono realizzati con uno standard pipetta estrattore (Es. Sutter P-97 pipette Puller) e vengono poi inseriti in tubi in plastica (Tygon, 1 / 16 &quot;) collegato a una fonte di vuoto. Questo sistema è utilizzato per aspirazione il grasso in eccesso attorno al tubo cuore. Forza di aspirazione è proporzionale al diametro della punta in modo pipette con le punte maggiori di 40 micron di diametro non deve essere utilizzato. Estrema cura deve essere presa per evitare di toccare il cuore stesso e perché la parte posteriore del cuore è molto fragile, che la regione dovrebbe essere evitato del tutto. </li><li> L&#39;adulto tubo cuore Drosophila è ora esposta e va a battere. Se necessario, il cuore della cuticola e allegata possono essere riposizionati da sollevando delicatamente la cuticola fuori la vaselina e con attenzione pigiatura indietro, sempre evitando ogni contatto con il tessuto cardiaco. Til suo punto la soluzione balneari la preparazione deve essere sostituita con AHL fresca. Questa preparazione deve essere lasciato equilibrare per 20-30 minuti con l&#39;ossigenazione prima di qualsiasi manipolazione. Se la preparazione deve essere mantenuta per più di 60 minuti dovrebbe essere perfusi con AHL fresca. </li></ol> Immobilizzare le larve di Drosophila per la registrazione ottica <ol><li> Mettere una goccia di colla Histoacryl vicino a ogni angolo del coprioggetto Sylgard. Aggiungere 50μl di B &amp; B buffer per le gocce di colla che si indurisce immediatamente. Riempire lo spazio tra i punti incollati con B &amp; B - la colla non mancherà di tenere il tampone sul vetrino. </li><li> Prendete una larva L3 errante e mettetelo su un foglio di carta velina bagnata in ghiaccio per 2 minuti. Questo rallenterà il suo movimento. Poi trasferire la larva nel buffer e orientarlo con la parte dorsale verso l&#39;alto (questo è il lato della larva che non ha le cinture dentino). Applicare rapidamente po &#39;di colla tra la regione della testa e del Sylgard. La colla si indurisce molto rapidamente e prevenire il capo della larva di muoversi. </li><li> Utilizzando pinze attentamente afferrare l&#39;animale dal spiracoli posteriori e delicatamente stretch. Fissare la larva in questa posizione applicando la colla lungo entrambi i lati della larva a metà posteriore. Per ridurre ulteriormente il movimento della cuticola dalla muscoli della parete del corpo, aggiungere la colla per i fianchi dell&#39;animale. La larva è ora fissato in questa posizione. Il coprioggetto può essere collocato in una piccola scatola di Petri piena di AHL o PBS. Battiti cardiaci possono essere registrati senza interferenze da parte dei movimenti parete del corpo. </li></ol> Rappresentante dei risultati: Il semi-intatti cuore adulto Drosophila batterà ritmicamente per ore seguenti dissezione quando mantenuto in fresco, AH ossigenata (vedi Dati supplementari, Ocorr, et al., 2007). Un esempio rappresentativo è mostrato in Movie 1. Cuori da 3 larva ° instar e vola giovani (1 - 3 settimane dopo eclosion) in genere mostrano un ritmo regolare cuore a tassi tra l&#39;1 - 3 Hz. Vola più di 3 settimane in genere sono più aritmici, tuttavia, questi cuori sono ancora in grado di battere spontaneamente per ore in AHL ossigenato. Cuori in mosche adulte che sono danneggiati come conseguenza della procedura di dissezione mostrano tipicamente le regioni localizzate di costrizione estrema, che non sono in grado di rilassarsi. Tassi di battito cardiaco più lento di 1 Hz sono raramente osservati nelle mosche meno di 3 settimane di età, di conseguenza tali preparati sono considerati danneggiati e vengono scartati. <a href="http://www.jove.com/files/ftp_upload/1425/MOVIE1.avi">Clicca qui per scaricare film 1.</a> - Una settimana quella vecchia volare tipo selvatico adulto (ceppo di laboratorio w 1118) che mostra il tubo esposto cuore addominale (anteriore a sinistra). Nota regolare, contrazioni ritmiche. Regioni opache nell&#39;angolo in alto a destra ci sono i restanti corpi grasso addominale; cellule rotonde su entrambi i lati del cuore sono cellule del pericardio.

<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>

Il modello Drosophila ha dimostrato di essere un potente strumento genetico che è stato usato per affrontare una serie di quesiti scientifici che spaziano dallo sviluppo embrionale all&#39;apprendimento e alla memoria. Di recente questo organismo modello versatile è stato utilizzato per studiare la genetica della funzione cardiaca in tempo reale. Un certo numero di tentativi di quantificare fisiologia cardiaca in adulti Drosophila si sono basati sulle osservazioni fatte nei moscerini intatta attraverso la cuticola add...

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<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>
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<td>Micro Dissecting Spring Scissors (straight)</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5620</td>
<td>&nbsp;</td>
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<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>
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<td>Glass Capillaries, 100ul</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>Glass Capillaries, fine</td>
<td>&nbsp;</td>
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<td>GB100T8P</td>
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<td>Pipette Puller</td>
<td>&nbsp;</td>
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<td>Both horizontal and vertical pipette pullers will work</td>
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<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>
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<td>Plastic tubing</td>
<td>&nbsp;</td>
<td>Tygon R-3603</td>
<td>1/32&#x201D; inner diameter</td>
<td>for small capillaries</td>
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<td>Histoacryl&reg; tissue adhesive</td>
<td>&nbsp;</td>
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visualizing the beating heart in drosophila

Il<em&gt; Drosophila</emCuore&gt; è recentemente emerso come un sistema di buon modello per l&#39;esame dei meccanismi genetici, cellulari e molecolari alla base della funzione nei cuori miogenico. Un passo fondamentale per esaminare la funzione cardiaca in tempo reale è trovare un modo per accedere al cuore in un modo che conserva la sua funzione miogenici pur consentendo l&#39;organo cuore pulsante di essere osservato e registrato. Due diversi metodi per osservare e registrare il cuore pulsante in entrambe le larve e adulti<em&gt; Drosophila</em&gt; Sono descritte qui. Il nostro semi-intatta la preparazione con le mosche adulte consente la visualizzazione chiara del cuore addominali senza interferenze da parte cuticola pigmentate e sovrastanti corpi grassi. Per registrare il cuore batte larvale è necessario immobilizzare la larva, che riduce al minimo i movimenti parete del corpo in modo da ridurre i movimenti cuore che non sono associati a contrazioni del miocardio. Le nostre metodologie di produzione stabile adulti e preparati cuore larvale che può battere per ore a tassi di Hz 1-3.

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

Visualizing the Beating Heart in Drosophila

Tecnica necessaria per visualizzare il cuore pulsante di larve e adulti<em&gt; Drosophila</em&gt; Sono presentati. Ogni fase della vita richiede una metodologia diversa.

Visualizzare il cuore pulsante di 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 Views.  The Sanford Burnham Institute for Medical Research. Tecnica necessaria per visualizzare il cuore pulsante di larve e adulti Drosophila Sono presentati. Ogni fase della vita richiede una metodologia diversa.

Video: 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.

Visualizzazione di Live Drosophila Gliali-giunzione neuromuscolare con tinture fluorescenti

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

Ricerca

Biologia

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.

Etichette fluorescenti di Drosophila Cuore Strutture

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.

Il monitoraggio delle funzioni cardiaca in larvali Drosophila melanogaster Per gli Studi fisiologici

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.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

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 &#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.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

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 &#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.

ΓH2AX quantificazione dei Foci, a campioni di tessuto

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 &#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.

Valutazione della distribuzione spaziale delle γH2AX seguenti radiazioni ionizzanti

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.

Clonogenica saggio: cellule aderenti

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.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

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.

Valutazione del calcio Sparks in intatte fibre muscolari scheletriche

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