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. circolazione Concentrazione hemocyte <ol><li> Per ottenere larve di circa la stessa fase di sviluppo per questa analisi, limitare la raccolta delle uova da garantire che le femmine di deporre le uova per un periodo di tempo fisso di 2-6 ore. </li><li> Raccogliere le larve nel sezionare pozzi piatto pieno di 1x tampone fosfato (PBS, Tabella 1). </li><li> Per ogni larva, mettere 10 microlitri 1x PBS in una provetta su ghiaccio e 10 microlitri 1x PBS su un rilievo da dissezione pulito. Posizionare il ...

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Su alterazione genetica o ambientale, i quattro metodi descritti qui possono essere utilizzati singolarmente o in combinazione di analizzare i processi distinti durante emopoiesi, come la segnalazione, la sopravvivenza, la proliferazione e la differenziazione Drosophila emopoiesi è un processo dinamico.; il numero di hemocytes per animale aumenta 35 e la struttura e l&#39;espressione genica della ghiandola linfatica variazioni 32 durante lo sviluppo. Prima di eseguire questi test, quindi,...

I complessi meccanismi che regolano i fattori di trascrizione e vie di segnalazione che coordinano lo sviluppo del sistema ematopoietico e che malfunzionamento malattie ematologiche sono ancora poco chiare. Questi fattori di trascrizione e vie di segnalazione, così come la loro regolamentazione, sono altamente conservati tra Drosophila e mammiferi emopoiesi 1-5. Così il sistema ematopoietico Drosophila rappresenta un ottimo modello genetico per definire i meccanismi molecolari che controllano l&#39;emopoiesi e malattie ematologiche sottostanti. Simile a mammiferi, Drosophila generare cellule del sangue, chiamate ematociti concentrati, in spazialmente e temporalmente distinte fasi di emopoiesi. Tradizionalmente, Drosophila emopoiesi è stato pensato per essere limitato alle fasi del mesoderma embrionale e nella ghiandola linfatica larvale. Recenti studi dimostrano che emopoiesi si verifica anche in larvale clu sessilisters e nell&#39;adulto addome 6-8. Tutte le fasi ematopoietiche producono due tipi di ematociti concentrati mature: plasmatocytes e cellule di cristallo. Plasmatocytes sono cellule macrofagi come coinvolti nella fagocitosi, l&#39;immunità innata, e la guarigione della ferita. celle a cristalli contengono pro-phenoloxidases necessari per melanizzazione, una reazione usato in risposte immunitarie insetti e la guarigione delle ferite. Emopoiesi larvale può generare un terzo tipo hemocyte maturo, chiamato lamellocyte, in risposta ad alcune sfide immunitario come parassitoide vespa infezione 9,10. Lamellocytes sono grandi, cellule aderenti che funzionano, in collaborazione con plasmatocytes e le cellule di cristallo, per incapsulare e neutralizzare le uova di vespa di cui in Drosophila larve. In assenza di parassitizzazione, lamellocytes non si trovano nel wild-type larve. masse melanotic assomigliano melanized, uova vespa incapsulati; molti ceppi mutanti di Drosophila sviluppano masse melanotici in assenza di parassitizzazione. La presenza di Lamellociti e / o masse melanotici possono essere indicativi di anomalie ematopoietiche. In realtà, il fenotipo di massa melanotico è stato utilizzato per identificare i geni e meccanismi coinvolti nella emopoiesi 11-14. Il sistema ematopoietico larvale è il più ampiamente studiato fino ad oggi. Si compone di ematociti concentrati circolanti nel emolinfa, i cluster hemocyte sessili fantasia sotto la cuticola, e ematociti concentrati che risiedono nella ghiandola linfatica. La ghiandola linfatica è una serie di lobi bilaterali collegati al vaso dorsale. Ogni lobo principale della ghiandola linfatica è diviso in tre zone principali. La zona più esterna è conosciuta come la zona corticale e contiene maturazione hemocytes. La zona più interna viene chiamata zona midollare e comprende precursori hemocyte quiescenti. La terza zona, la segnalazione posteriori centro, è un piccolo gruppo di cellule alla base della ghiandola linfa che agiscono come una cellula simile nicchia stelo. I primi lavori stabilito funzioni critiche per Notch 15-18 </sup&gt;, Riccio 19,20, JAK-STAT 18 e ali 21 attività per regolare lo sviluppo della ghiandola linfa larvale. Studi più recenti hanno dimostrato che BMP 22, FGF-Ras 23, e Hippo 24,25 funzione di segnalazione anche all&#39;interno della ghiandola linfatica larvale. Quattro saggi ematopoietiche larvali qui esposti descrivono 1) misura la concentrazione hemocyte circolazione, definito come numero di cellule per unità di volume, 2) l&#39;isolamento e il fissaggio di circolazione hemocytes per immunoistochimica, 3) la visualizzazione di cellule di cristallo in vivo, e 4) dissezione, il fissaggio e il montaggio ghiandole linfatiche per immunoistochimica. Questi test possono essere utilizzati come letture ematopoietiche per valutare le funzioni ei regolamenti dei percorsi di segnalazione nel sistema ematopoietico larvale. Mentre questi metodi sono stati utilizzati in precedenza nel campo, documentazione visiva di questi saggi ha iniziato solo recentemente 8,26-30. Diverse pubblicazioni citate qui ci sono di aiutorisorse ful che descrivono metodi simili e marcatori ematopoietici 26,31-33. Inoltre, Trol e Viking sono marcatori utili della membrana linfatico della ghiandola seminterrato.

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	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">p200 pipette</td>
			<td style="width:176px;">Eppendorf</td>
			<td>3120000054</td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<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

Molti paralleli esistenti tra i sistemi ematopoietiche mammiferi Drosophila e, anche se Drosophila manca il lignaggio linfoide che caratterizzano mammiferi immunità adattativa. Drosophila e mammiferi emopoiesi si verificano in spazialmente e temporalmente distinte fasi per la produzione di diverse linee cellulari del sangue. Entrambi i sistemi mantengono riserve di progenitori di cellule del sangue con il quale espandere o sostituire linee maturi. Il sistema ematopoietico permette Drosophila e mammiferi per rispondere e di adattarsi alle sfide del sistema immunitario. È importante sottolineare che i regolatori trascrizionali e vie di segnalazione che controllano la generazione, la manutenzione, e la funzione del sistema ematopoietico sono conservati dalle mosche ai mammiferi. Queste somiglianze consentono Drosophila da utilizzare per geneticamente modello di sviluppo ematopoietiche e la malattia. Qui dettaglio test per esaminare il sistema ematopoietico di Drosophila larve. Inparticolare, si delineano i metodi per misurare il numero di cellule del sangue e la concentrazione, visualizzare una specifica stirpe maturo in vivo, ed eseguire immunoistochimica sulle cellule del sangue in circolazione e nell&#39;organo ematopoietiche. Questi test possono rivelare cambiamenti nell&#39;espressione genica e dei processi cellulari tra cui la segnalazione, la sopravvivenza, la proliferazione e la differenziazione e può essere utilizzato per indagare su una serie di domande riguardanti emopoiesi. In combinazione con gli strumenti genetici disponibili in Drosophila, questi test possono essere utilizzati per valutare il sistema ematopoietico su alterazioni genetiche definiti. Sebbene non specificamente qui delineato, questi saggi possono anche essere utilizzati per esaminare l&#39;effetto di alterazioni ambientali, come infezioni o dieta, sul sistema ematopoietico.

Di circolazione hemocyte Concentrazione Numeri hemocyte aumentano durante lo sviluppo larvale 35. Per illustrare che questo metodo rileva le differenze nei numeri hemocyte e concentrazione, indipendentemente dalla causa biologica, abbiamo misurato concentrazioni hemocyte di ritardo e non ritardata larve. Perdita di ormone prothoracicotropic (ptth) per l&#39;ablazione genetica dei neuroni ptth produttrici (ptth&gt; cupo...

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

Sistemi ematopoietiche mammiferi Drosophila e condividono molte caratteristiche comuni, rendendo Drosophila un modello genetico interessante per lo studio ematopoiesi. Qui mostriamo dissezione e montaggio del grande organo ematopoietico larvale per immunoistochimica. Descriviamo anche metodi per saggiare i vari compartimenti ematopoietici larvali tra emociti circolanti e le cellule di cristallo sessili.

I metodi per esaminare le ghiandole linfatiche e ematociti concentrati in<em&gt; Drosophila</em&gt; Le larve

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

Analisi delle larve Zebrafish muscolo scheletrico Integrità con Blu di Evans 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 ...

Ricerca

Biologia dello sviluppo

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

Dissezione e macchiatura immunofluorescente del corpo di fungo e del fotoricettore neuroni nel cervello adulto 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

Metodi per pH intracellulare Imaging del lignaggio di cellule staminali del follicolo in Live tessuto ovarico di Drosophila

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

Cella di aggregazione saggi per valutare l'associazione della tacca della drosofila con Trans-ligandi e la sua inibizione di Cis-ligandi

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

Metodi per lo studio della rigenerazione in 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

Scongelamento, coltura e Cryopreserving linee di cellule di Drosophila

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

Metodi per testare la disgregazione endocrina nella Drosophila 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

Intero Monte Immunohistochimica in Embrioni e Larve di pesce zebra

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

Rimozione dell'occhio nelle larve di zebrafish viventi per esaminare la crescita dipendente dall'innervazione e lo sviluppo del sistema visivo

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