Name: live imaging of apoptotic cell clearance during drosophila embryogenesis
Uploaded: 2013-08-18T14:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis at JoVE.com

Dette arbejde blev støttet af et Marie Curie reintegrationsstipendium (IRG249084). Vi takker alle medlemmer af Kurant laboratorium.

At følge dynamikken i apoptotic celle clearance in vivo to populationer af celler skal mærkes: fagocytceller og apoptotiske celler. For at markere fagocytiske cellepopulationer vi bruger en Drosophila linje, der indeholder simulere cytGFP markør, hvilke etiketter udelukkende fagocytceller i embryo: makrofager, glia og ectoderm 8.. Man kan bruge forskellige markører for fagocytceller, herunder linjer, der indeholder en hemocyte-specifik Gal4 driver <e...

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Apoptotisk celle clearance er en kritisk sidste stadie af apoptose, som er meget dynamisk. Derfor realtid undersøgelser af processen er af allerstørste betydning. Her beskriver vi en protokol, der muliggør overvågning af apoptotic celle clearance i levende udviklingslandene Drosophila embryoner. I denne procedure, skal to populationer af celler mærkes med specifikke markører: apoptotiske celler og fagocytter. Den simu-cytGFP reporter er egnet til overvågning fagocytiske makrofager, glia og ectod...

Den rette fjernelse af uønskede eller afvigende celler gennem apoptose og efterfølgende fagocytose er afgørende for embryonale udvikling samt vævshomeostase i den voksne. Fagocytose af apoptotiske celler eller apoptotisk celle clearance er en meget dynamisk proces, der forløber i fire trin: (1) ansættelse af fagocytter til apoptotiske celle (&#39;find-mig &quot;), (2) anerkendelse af cellen som et mål for fagocytose ( &#39;spise mig &quot;), og (3) engulfment, efterfulgt af (4) fagosomet modning og nedbrydning af apoptotiske partikel 1-5. Der er to typer af fagocytter: &#39;professionelle&#39; makrofager og umodne dendritiske celler, og &quot;ikke-professionelle&quot; tissue-hjemmehørende omkringliggende celler, som er afgørende for apoptotic celle clearance under metazoan udvikling 6,7. I Drosophila udvikling, sker fjernelsen af overflødige celler gennem apoptose i tre main faser, først i midten til slutningen af ​​embryo, og derefter i midten puppe, og igen i den tidlige voksen. Under embryogenese apoptotiske partikler fjernes ved &#39;professionelle&#39; fagocytter, makrofager, og af den &quot;ikke-professionelle&quot; ectoderm og glia 8,9. I vores tidligere arbejde har vi identificeret en fagocytisk receptor, Six-mikron-under (SIMU), som udelukkende udtrykkes i fagocyterende celler under embryogenese. Brug af simu promotor vi genereret en specifik markør for fagocytiske cellepopulationer (simulere cytGFP), som gør os i stand til at overvåge makrofager, ectoderm og glia samtidig i en live embryo under udvikling 8.. Derudover, ved anvendelse af specifikke markører for apoptotiske celler i forskellige stadier af apoptose vi er i stand til at følge dynamikken i apoptotisk celle clearance in vivo.

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 <td>Reagent</td>
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 <td>Annexin V </td>
 <td>Molecular Probes</td>
 <td>A35108</td>
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 <td>PhiPhiLux G2D2</td>
 <td>OncoImmunin</td>
 <td>A304R2G-5</td>
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 <td>LysoTracker</td>
 <td>Molecular Probes</td>
 <td>L-7528</td>
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 <td>Halocarbon oil 700</td>
 <td>Sigma</td>
 <td>H8898</td>
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 <td>Capillary tubing</td>
 <td>FHC </td>
 <td>30-30-0</td>
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 <td>Cell Strainer</td>
 <td>SPL</td>
 <td>93100</td>
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 <td>Paintbrush</td>
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live imaging of apoptotic cell clearance during drosophila embryogenesis

Den rette fjernelse af uønskede eller afvigende celler gennem apoptose og efterfølgende fagocytose (apoptotic celle clearance) er afgørende for en normal udvikling i alle metazoiske organismer. Apoptotisk celle clearance er en meget dynamisk proces intimt forbundet med celledød, unengulfed apoptotiske celler er næsten ikke ses in vivo under normale forhold. For at forstå de forskellige trin i apoptotic celle clearance og sammenligne &#39;professionelle&#39; fagocytter - makrofager og dendritiske celler til &quot;ikke-professionelle&quot; - tissue-hjemmehørende omkringliggende celler, in vivo billeddannelse af processen er yderst værdifuld. Her beskriver vi en protokol for at studere apoptotic celle clearance i levende Drosophila embryoner. At følge dynamikken i forskellige trin i fagocytose vi bruger specifikke markører for apoptotiske celler og fagocytter. Derudover kan vi overvåge to fagocyt systemer i parallel: &#39;professionelle&#39; makrofager og &#39;semi-professional &#39;glia i udviklingslandene centralnervesystemet (CNS). Den her beskrevne metode anvender Drosophila embryo som en fremragende model for tidstro studier af apoptotic celle clearance.

Repræsentative billeder fra en film i hvilken apoptotiske celler er mærket med Annexin V, og glia, ektoderm og makrofager er mærket med simule-cytGFP er vist i figur 2.. Hver ramme er en fremskrivning af 3 skiver 2 um hver. Den embryo af etape 15 er rigtigt placeret viser embryonale CNS i midten med godt mærket glia (g). Makrofager (m), som for det meste udenfor CNS, udviser stærk cytoplasmatisk GFP-ekspression. Ectodermal celler er også mærket med cytoplasma...

Watch this Scientific Journal Video about Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis at JoVE.com

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Her beskriver vi en effektiv metode til at studere dynamikken i apoptotisk celle clearance<em&gt; In vivo</em&gt;. Denne fremgangsmåde anvender leve<em&gt; Drosophila</em&gt; Embryoner som en stærk model for overvågning fagocytose af apoptotiske celler under anvendelse af specifikke mærkning af apoptotiske celler og fagocytter.

Levende Imaging af Apoptotisk Cell Clearance under<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  ...

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Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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