Name: methods to examine the lymph gland and hemocytes in drosophila larvae
Uploaded: 2016-11-28T14:00:00
Description: Watch this Scientific Journal Video about Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae at JoVE.com

We thank Matthew O'Connell, Maryam Jahanshahi, and Andreas Jenny for assistance. We thank Istv&#225;n And&#243; for plasmatocyte-specific antibodies, Utpal Banerjee for dome-meso-EBFP2 flies, Julian Martinez-Agosto for antp&gt;GFP flies, and Michael O'Connor for ptth and ptth&gt;grim flies. These methods were developed with support by the Kimmel Foundation, the Leukemia &amp; Lymphoma Society, NIH/NCI R01CA140451, NSF 1257939, DOD/NFRP W81XWH-14-1-0059, and NIH/NCI T32CA078207.

1. Circulerende hemocyte Concentratie <ol><li> Om larven van ruwweg hetzelfde ontwikkelingsstadium te krijgen voor deze test beperken eierverzameling doordat vrouwtjes om eieren te leggen voor een vaste periode van 2-6 uur. </li><li> Verzamel larven ontleden schotel putjes gevuld met 1x fosfaat gebufferde zoutoplossing (PBS, tabel 1). </li><li> Voor elke larve, plaats 10 pi 1x PBS in een microcentrifugebuis op ijs en 10 pi 1x PBS op een schone dissectie pad. Plaats de dissectie pad op een verlichte st...

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Bij genetische of ecologische verandering kunnen de vier methoden beschreven afzonderlijk of samen worden gebruikt om verschillende processen tijdens hematopoiese zoals signalering, overleving, proliferatie, differentiatie en hematopoëse analyseren Drosophila is een dynamisch proces.; het aantal hemocytes per dier verhoogt 35 de en genexpressie van de lymfeklier 32 verandert tijdens de ontwikkeling. Voorafgaand aan het uitvoeren van deze testen, daarom is het essentieel om eieren verzamel...

De complexe mechanismen reguleren van de transcriptiefactoren en signaalroutes die de ontwikkeling van het hematopoietische systeem dat storingen in hematologische ziekten coördineren blijven slecht begrepen. Deze transcriptiefactoren signaalbanen, en hun regulatie, zijn sterk geconserveerd tussen Drosophila en zoogdieren hematopoiese 1-5. Dus de Drosophila hematopoëtische systeem vormt een uitstekende genetisch model om de moleculaire mechanismen die hematopoiese en onderliggende hematologische ziektes bepalen. Soortgelijke zoogdieren, Drosophila genereren bloedcellen, genaamd hemocytes in ruimte en tijd verschillende fasen van hematopoiese. Traditioneel werd gedacht Drosophila hematopoiese te beperken tot fasen in de embryonale mesoderm en het larvale lymfeklier. Recente studies bewijzen dat hematopoiese komt ook voor in larvale wintereik clusters en in de volwassen buik 6-8. Alle hematopoietische fasen produceren twee soorten volwassen hemocytes: plasmatocytes en kristal cellen. Plasmatocytes zijn macrofaag-achtige cellen betrokken bij fagocytose, aangeboren immuniteit en wondgenezing. Kristalcellen bevatten pro-fenoloxidasen nodig melanisatie, een reactie die in insectencellen immuunresponsen en wondgenezing. Larvale hematopoiese kan een derde volwassen hemocyte soort genereren, een zogenaamde lamellocyte, als reactie op bepaalde immuun uitdagingen, zoals de sluipwesp infectie 9,10. Lamellocytes zijn groot, hechtende cellen die functioneren in samenhang met plasmatocytes en kristal cellen, te integreren en te neutraliseren gelegd in Drosophila larven wesp eieren. Aangezien parasitering worden lamellocytes niet gevonden in wildtype larven. Melanotisch massa&#39;s lijken gemelamineerde, ingekapselde wesp eieren; vele Drosophila mutant stammen ontwikkelen melanotisch massa in afwezigheid van parasitering. De aanwezigheid van Lamellocyten en / of melanotisch massa&#39;s kan een indicatie van hematopoietische afwijkingen zijn. In feite heeft de melanotisch massa fenotype gebruikt om genen en routes die betrokken zijn bij hematopoiese 11-14 identificeren. Het larvale hematopoietische systeem is het meest uitgebreid bestudeerd tot nu toe. Het bestaat uit hemocytes circuleert in de hemolymfe, zittend hemocyte clusters gevormd onder de opperhuid en hemocytes die in de lymfeklier. De lymfeklier is een reeks bilaterale lobben aan de dorsale vat. Elke primaire kwab van de lymfeklier is verdeeld in drie belangrijke zones. De buitenste zone staat bekend als de corticale zone en bevat rijpen hemocytes. De binnenste zone wordt het medullaire zone genoemd en bestaat uit zwakke hemocyte precursors. De derde zone, het achterste signalering centrum, is een kleine groep cellen aan de basis van de lymfeklier die fungeren als stamcelachtige niche. Vroeg werk opgericht kritische functies voor Notch 15-18 </sup&gt;, Hedgehog 19,20, JAK-STAT 18 en Vleugelloze 21 activiteit larvale lymfeklier ontwikkeling reguleren. Meer recente studies hebben aangetoond dat 22 BMP, FGF-Ras 23 en Hippo 24.25 signalering ook functioneert binnen de larvale lymfeklier. Vier larvale hematopoietische assays geschetste beschrijven 1) het meten van circulerende hemocyte concentratie, gedefinieerd als het aantal cellen per volume-eenheid, 2) het isoleren en fixeren circulerende hemocytes voor immunohistochemie, 3) visualiseren crystal cellen in vivo, en 4) het ontleden, het bevestigen en mounting lymfeklieren voor immunohistochemie. Deze testen kunnen worden gebruikt als hematopoietische uitlezingen de functies en voorschriften van signaaltransductiewegen in het larvale hematopoietische systeem te beoordelen. Hoewel deze werkwijzen die eerder in het veld zijn gebruikt, is beelddocumentatie van deze testen pas onlangs begonnen 8,26-30. Verschillende publicaties hier aangehaald zijn hulpful middelen beschrijven gelijkaardige methodes en hematopoietische markers 26,31-33. Bovendien, Trol en Viking zijn nuttige markers van de lymfeklier basaal membraan.

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			<td height="21" style="height:21px;width:229px;">PBS tablets</td>
			<td style="width:176px;">MP Biomedicals</td>
			<td style="width:163px;">2810305</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">dissecting dish</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">7220-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">microcentrifuge tube</td>
			<td style="width:176px;">Denville</td>
			<td style="width:163px;">C2170</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:229px;">silicone dissecting pad, made from Sylgard 184 kit</td>
			<td style="width:176px;">Krayden (distributed through Fisher)</td>
			<td style="width:163px;">NC9644388 (Fisher catalog number)</td>
			<td style="width:236px;">Made in petri dish by mixing components of Sylgard elastomer kit according to manufacturer instructions.</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:229px;">stereomicroscope</td>
			<td style="width:176px;">Morrell Instruments (Nikon distributor)</td>
			<td>mna42000, mma36300</td>
			<td>Nikon models SMZ1000 and SMZ645</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">tissue wipe</td>
			<td style="width:176px;">VWR</td>
			<td style="width:163px;">82003-820</td>
			<td></td>
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			<td height="42" style="height:42px;width:229px;">forceps</td>
			<td style="width:176px;">Electron Microscopy Sciences</td>
			<td style="width:163px;">72700-DZ</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">p200 pipette</td>
			<td style="width:176px;">Eppendorf</td>
			<td>3120000054</td>
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			<td height="42" style="height:42px;width:229px;">Countess Automated Cell Counter</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:163px;">C10227</td>
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			<td height="42" style="height:42px;width:229px;">Countess cell counting chamber slides</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:163px;">C10283</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:229px;">hemocytometer</td>
			<td style="width:176px;">Hausser Scientific</td>
			<td style="width:163px;">3200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">trypan blue stain</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:163px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">formaldehyde</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">BP531-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Triton</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">BP151-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tween 20</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">BP337-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">bovine serum albumin</td>
			<td style="width:176px;">Rocky Mountain Biologicals</td>
			<td style="width:163px;">BSA-BSH-01K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">normal goat serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:163px;">G9023-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">normal donkey serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:163px;">D9663-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">200 proof ethanol</td>
			<td style="width:176px;">VWR</td>
			<td style="width:163px;">V1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">N-propyl gallate</td>
			<td style="width:176px;">MP Biomedicals</td>
			<td style="width:163px;">102747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">glycerol</td>
			<td style="width:176px;">VWR</td>
			<td style="width:163px;">EM-4750</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">DAPI (4&#8217;,6-diamidino-2-phenylindole)</td>
			<td style="width:176px;">Fisher</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">6-well plate</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">12-well plate</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">351143</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">microscope cover glass, 22 mm square</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">12-544-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">microscope cover glass, 18mm circular</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">12-545-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">glass microscope slides</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">22-034-980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">thermal cycler</td>
			<td style="width:176px;">Eppendorf</td>
			<td>E950010037</td>
			<td>Mastercycler EP Gradient S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">PCR tubes</td>
			<td style="width:176px;">USA Scientific</td>
			<td style="width:163px;">1402-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">24-well plate</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">351147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">disposable transfer pipet</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">13-711-9AM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">fluorescence microscope</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:163px;"></td>
			<td>Axio Imager.Z1</td>
		</tr>
	</tbody>
</table>

methods to examine the lymph gland and hemocytes in drosophila larvae

Vele parallellen tussen de Drosophila en zoogdierlijke bloedvormende organen, hoewel niet de Drosophila lymfoïde oorsprong die zoogdieren adaptieve immuniteit karakteriseren. Drosophila en zoogdieren hematopoiese voorkomen in ruimte en tijd verschillende fasen verschillende bloedcellijnen produceren. Beide systemen onderhouden reservoirs van bloedcel voorlopers waarmee uit te breiden of te vervangen volwassen geslachten. Het hematopoietische systeem maakt Drosophila en zoogdieren te reageren op en aan te passen aan immune uitdagingen. Belangrijk is dat de transcriptie regulatoren en signaalwegen dat de productie, het onderhoud, en de functie van het hematopoietische systeem te bedienen zijn geconserveerd uit vliegt naar zoogdieren. Deze overeenkomsten laten Drosophila worden gebruikt om genetisch model hematopoietische ontwikkeling en ziekte. Hier hebben we detail assays voor het hematopoietische systeem van Drosophila larven te onderzoeken. InVooral schetsen we methoden om bloedcellen nummers en concentratie te meten, visualiseren een specifiek volwassen afkomst in vivo, en het uitvoeren van immunohistochemie op bloedcellen in omloop en in de hematopoietische orgel. Deze testen kunnen veranderingen in genexpressie en cellulaire processen waaronder signalering, overleving, proliferatie en differentiatie zichtbaar en kan worden gebruikt om een ​​verscheidenheid van vragen met betrekking hematopoiese onderzoeken. Gecombineerd met de genetische hulpmiddelen in Drosophila, kunnen deze assays worden toegepast om het hematopoëtische systeem van gedefinieerde genetische veranderingen te evalueren. Hoewel niet specifiek hier beschreven, kunnen deze bepalingen ook worden gebruikt voor kwantificering van de veranderingen, zoals infectie of voeding, van het hematopoietische systeem te onderzoeken.

Circulerende hemocyte Concentratie Hemocyte aantallen toenemen gedurende larvale ontwikkeling 35. Om te illustreren dat deze methode detecteert verschillen hemocyte aantallen en concentratie, ongeacht de biologische oorzaak, maten we hemocyte concentraties van vertraagde en niet-vertraagde larven. Verlies van prothoracicotropic hormoon (ptth) door genetische ablatie van ptth producerende neuronen (ptth&gt; grim) produc...

Watch this Scientific Journal Video about Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae at JoVE.com

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila en zoogdieren hematopoietische systemen hebben veel gemeenschappelijke kenmerken, waardoor Drosophila een aantrekkelijke genetische model om hematopoiese te bestuderen. Hier laten we dissectie en montage van grote larvale hematopoëtische organen voor immunohistochemie. We beschrijven ook werkwijzen om verschillende larvale hematopoietische compartimenten waaronder circulerende hemocytes sessiele kristalcellen testen.

Methoden om Onderzoek de lymfeklier en Hemocytes in<em&gt; Drosophila</em&gt; Larven

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Research

JoVE Journal

Developmental Biology

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Analyse van de zebravis Larven skeletspieren Integriteit met Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissectie en immunefluorescentie kleuring van Mushroom lichaam en fotoreceptor neuronen in de hersenen van de volwassen Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Methoden voor Imaging intracellulaire pH van de follikel stamcel overdrachtslijn in Live Drosophila eierstokweefsel

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cel aggregatie testen om te evalueren van de Binding van de Drosophila inkeping met Trans-liganden en haar remming door Cis-liganden

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Methods for the Study of Regeneration in Stentor

Methods for the Study of Regeneration in Stentor

Methoden voor de studie van regeneratie in de Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Ontdooien, kweken en Cryopreserving van Drosophila cellijnen

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

Methods to Test Endocrine Disruption in Drosophila melanogaster

Methoden voor het testen van endocriene verstoring in fruitvliegje melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Hele mount immunohistochemie in zebravissen embryo's en larven

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Oogverwijdering bij levende zebravislarven om innervatie-afhankelijke groei en ontwikkeling van het visuele systeem te onderzoeken

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...