Name: live imaging of drosophila melanogaster embryonic hemocyte migrations
Uploaded: 2010-02-12T18:30:00
Description: Watch this Scientific Journal Video about Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations at JoVE.com

Questo protocollo è stato sviluppato attraverso il nostro lavoro all&#39;interno e in collaborazione con i laboratori di Paul Martin e Antonio Jacinto. Ringraziamo il Centro archivi Bloomington per il suo servizio eccellente e la comunità Drosophila per continuare a condividere linee di volare. BS è attualmente finanziata da una sovvenzione BBSRC progetto. WW è finanziato da una borsa di studio Career Development Wellcome Trust.

Preparazione <ol><li> Ottenere adeguate linee di Drosophila contenente un hemocyte specifico GAL4 driver (ad esempio SRP-GAL4 2) e un giornalista geneticamente codificato fluorescenti SUP sotto controllo (ad esempio UAS-GFP). Vola omozigoti per SRP-GAL4, UAS-GMA 3 o CRQ-GAL4, UAS-GFP 4, 5 sono particolarmente utili per scopi di imaging (nb GMA è fuso con la GFP actina dominio di legame moesina), vedi sotto per una discussione di la gamma di...

<ol>
	<li>Brand, A. H., 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=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PDGF/VEGF+receptor+controls+blood+cell+survival+in+Drosophila.">The PDGF/VEGF receptor controls blood cell survival in Drosophila.</a> Dev Cell. 7, 73-84 (2004).</li><li>Dutta, D., Bloor, J. W., Ruiz-Gomez, M., VijayRaghavan, K., Kiehart, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+morphogenetic+movements+in+Drosophila+using+Gal4-UAS-driven+expression+of+GFP+fused+to+the+actin-binding+domain+of+moesin.">Real-time imaging of morphogenetic movements in Drosophila using Gal4-UAS-driven expression of GFP fused to the actin-binding domain of moesin.</a> Genesis. 34, 146-151 (2002).</li><li>Stramer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+wound+inflammation+in+Drosophila+embryos+reveals+key+roles+for+small+GTPases+during+in+vivo+cell+migration.">Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration.</a> J Cell Biol. 168, 567-573 (2005).</li><li>Wood, W., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+embryonic+haemocytes:+masters+of+multitasking.">Drosophila melanogaster embryonic haemocytes: masters of multitasking.</a> Nat Rev Mol Cell Biol. 8, 542-551 (2007).</li><li>Halfon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+protein+reporters+for+use+with+the+Drosophila+Gal4+expression+system+and+for+vital+detection+of+balancer+chromosomes.">New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes.</a> Genesis. 34, 135-138 (2002).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <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 protocols. , (2000).</li><li>Tepass, U., Fessler, L. I., Aziz, A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+of+hemocytes+and+their+relationship+to+cell+death+in+Drosophila.">Embryonic origin of hemocytes and their relationship to cell death in Drosophila.</a> Development. 120, 1829-1837 (1994).</li><li>Millard, T. H., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+filopodial+interactions+during+the+zippering+phase+of+Drosophila+dorsal+closure.">Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure.</a> Development. 135, 621-626 (2008).</li><li>Doerflinger, H., Benton, R., Shulman, J. M., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+PAR-1+in+regulating+the+polarised+microtubule+cytoskeleton+in+the+Drosophila+follicular+epithelium.">The role of PAR-1 in regulating the polarised microtubule cytoskeleton in the Drosophila follicular epithelium.</a> Development. 130, 3965-3975 (2003).</li><li>Olofsson, B., Page, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Condensation+of+the+central+nervous+system+in+embryonic+Drosophila+is+inhibited+by+blocking+hemocyte+migration+or+neural+activity.">Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity.</a> Dev Biol. 279, 233-243 (2005).</li><li>Paladi, M., Tepass, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+Rho+GTPases+in+embryonic+blood+cell+migration+in+Drosophila.">Function of Rho GTPases in embryonic blood cell migration in Drosophila.</a> J Cell Sci. 117, 6313-6326 (2004).</li><li>Vlisidou, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+embryos+as+model+systems+for+monitoring+bacterial+infection+in+real+time.">Drosophila embryos as model systems for monitoring bacterial infection in real time.</a> PLoS Pathog. 5, e1000518-e1000518 (2009).</li><li>Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+actin-based+epithelial+adhesion+and+cell+matching+during+Drosophila+dorsal+closure.">Dynamic actin-based epithelial adhesion and cell matching during Drosophila dorsal closure.</a> Curr Biol. 10, 1420-1426 (2000).</li><li>Wood, W., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cell+movement+during+dorsal+closure+in+Drosophila+embryos.">Imaging cell movement during dorsal closure in Drosophila embryos.</a> Methods Mol Biol. 294, 203-210 (2005).</li><li>Kunwar, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tre1+GPCR+initiates+germ+cell+transepithelial+migration+by+regulating+Drosophila+melanogaster+E-cadherin.">Tre1 GPCR initiates germ cell transepithelial migration by regulating Drosophila melanogaster E-cadherin.</a> J Cell Biol. 183, 157-168 (2008).</li></ol>

Gli elementi più importanti di questa procedura sono la selezione di embrioni sani con ematociti concentrati etichettati in modo chiaro e montare con cura senza danneggiarli. Una volta che gli embrioni sono l&#39;olio alocarburi sono resistenti alla disidratazione e una volta montata può essere ripreso per diverse ore. Nelle nostre mani possiamo ematociti concentrati immagine per tre ore, con disidratazione trascurabile dell&#39;embrione o del foto-danno evidente, prendendo un z-pila di immagini ogni tre minuti sul no...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>70&mu;m pores</td>
</tr>
<tr>
<td>Halcarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H8898</td>
<td>&nbsp;</td>
<tr>
<td>Lumox/Petriperm dish</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>96077305</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

live imaging of drosophila melanogaster embryonic hemocyte migrations

Molti studi sulle cellule migrazione indirizzo utilizzando<em&gt; In vitro</emMetodi&gt;, considerando che l&#39;ambiente fisiologicamente rilevanti è quello dell&#39;organismo stesso. Qui vi presentiamo un protocollo per il montaggio di<em&gt; Drosophila melanogaster</em&gt; Gli embrioni e le successive immagini dal vivo di ematociti concentrati fluorescente, i macrofagi embrionale di questo organismo. Utilizzando il sistema UAS-GAL4<sup&gt; 1</sup&gt; Guidiamo l&#39;espressione di una varietà di geneticamente codificato, marker fluorescente taggato in ematociti concentrati a seguire i loro dispersione di sviluppo in tutto l&#39;embrione. A seguito di raccolta di embrioni allo stadio di sviluppo desiderato, il corion esterno viene rimosso e gli embrioni vengono poi montati in olio alocarburi tra un idrofobo, gas-permeabile membrana e un coprioggetto di vetro per l&#39;imaging dal vivo. Oltre al lordo parametri migratori come la velocità e direzionalità, imaging ad alta risoluzione accoppiato con l&#39;uso di reporter fluorescenti di F-actina e microtubuli in grado di fornire informazioni più dettagliate circa la dinamica di questi componenti del citoscheletro.

Watch this Scientific Journal Video about Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations at JoVE.com

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Ematociti concentrati Drosophila disperdere sulla totalità dello sviluppo dell&#39;embrione. Questo protocollo dimostra come montare e immagine queste migrazioni uso di embrioni con ematociti concentrati fluorescente.

Immagini dal vivo di Drosophila melanogaster Migrazioni Hemocyte embrionali

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

Research

JoVE Journal

Biology

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Dissection of Larval CNS in Drosophila Melanogaster

Dissezione del sistema nervoso centrale larvali in Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Ricerca

Biologia

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

Vivere Imaging of the Brain Zebrafish embrionali da microscopia confocale

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 ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell imaging e analisi quantitativa della embrionali in cellule epiteliali Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Dissection of Oenocytes from Adult Drosophila melanogaster

Dissection of Oenocytes from Adult Drosophila melanogaster

Dissezione di Oenocytes da adulti Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

Isolation of Drosophila melanogaster Testes

Isolamento di Drosophila melanogaster Testicoli

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Preparing Individual Drosophila Egg Chambers for Live Imaging

Preparazione individuale Drosophila Egg Chambers per l&#39;imaging dal vivo

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 GFP-labeled Proteins in Drosophila Oocytes

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Vivo Imaging delle proteine ​​GFP-etichettati in<em&gt; Drosophila</em&gt; Gli oociti

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Measurement of Lifespan in Drosophila melanogaster

Measurement of Lifespan in Drosophila melanogaster

Misura della durata della vita in<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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  ...

Imaging Through the Pupal Case of Drosophila melanogaster

Imaging Through the Pupal Case of Drosophila melanogaster

Imaging attraverso il caso pupa di<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...