Questo lavoro è stato finanziato da una sovvenzione della BSF USA-Israele a BS e EDSBS è un incombente della cattedra Hilda e Cecil Lewis in Genetica Molecolare.

NOTA: Le procedure che coinvolgono soggetti animali sono stati approvati dalla cura e uso degli animali Comitato Istituzionale (IACUC) presso il Weizmann Institute of Science. Preparazione del campione 1 <ol><li> Isolamento di acini pancreatici <ol><li> Preparare 300 ml di fresca bicarbonato di Krebs-Ringer HEPES (KRBH) mezzo prima di iniziare. Mescolare 140 mM NaCl, 4.7 mM KCl, 1.16 mM MgCl 2, 1 mM CaCl 2, 11 Glucosio mM, HEPES 10 mM. Aggiungi albumina sierica bovina (BSA) e / ml inibitore 0,1 mg di tripsina di soia (STI) (media risospensione). Preparare 5-10 ml di risospensione medie integrate con collagenasi (~ 0,75 mg / ml) (media digestione). </li><li> Ossigenare il terreno KRB per ~ 30 min. Aggiungi BSA dopo ossigenazione per evitare di bubbling. Portare risospensione e digestione medium a 37 ° C. NOTA: ossigenante medio riduce la mortalità delle cellule e migliora la risposta ai secretagoghi. L&#39;effetto di ossigenazione risulta almeno per 6 ore. </li><li&gt; sacrificare il mouse per dislocazione cervicale o di CO 2 asfissia, secondo le linee guida per la cura degli animali istituzionali. Posizionare l&#39;animale sacrificato sulla sua schiena, allegarlo a un pad dissezione e pulire la superficie addominale con etanolo. NOTA: Per evitare contaminazioni, sterilizzare in autoclave gli strumenti di dissezione e l&#39;acqua utilizzata per KRBH medio prima dell&#39;uso. Filtrare KRBH mezzo attraverso un filtro da 0,2 micron. Prima di asportazione del pancreas sciacquare gli strumenti di dissezione con il 70% di etanolo e procedere dissezione su un banco pulito. Effettuare infezioni virali in un armadio livello 3 bio-sicurezza. Aggiungi antibiotici per l&#39;/ N mezzo di coltura O (vedere paragrafo 4). </li></ol></li><li> L&#39;asportazione del pancreas murino <ol><li> Incisione ampiamente la cute addominale e lo strato sottocutaneo. Identificare il fegato, stomaco, intestino, pancreas e milza (Figura 1). NOTA: Nelle seguenti procedure, gestire il tessuto pancreatico delicatamente ed evitare l&#39;applicazione di un eccessivo forc meccanicae per evitare l&#39;auto-digestione della morte dei tessuti e delle cellule. </li><li> Utilizzando due pinze smussate, tirare fuori il piccolo intestino. Inizia la piccola estremità prossimale dell&#39;intestino, vicino all&#39;uscita dallo stomaco. Separare il pancreas dall&#39;intestino e conservarlo all&#39;interno della cavità del corpo. Una volta raggiunto l&#39;intestino crasso, notare un tessuto connettivo rosa / bianco. Da non confondere con il pancreas. </li><li> Svuota il tessuto pancreatico quanto possibile senza la raccolta tessuto adiposo o connettivo. Rimuovere il pancreas staccando dalla milza e lo stomaco e il taglio dei vasi sanguigni addominali sotto di esso (Figura 1A-C). NOTA: Poiché il rendimento di acini pancreatici da un singolo animale è elevato, si può astenersi dal raccogliere tutto il tessuto pancreatico per impedire la raccolta di altri tessuti. </li></ol></li><li> Collagenase digestione <ol><li> Sciacquare il pancreas un paio di volte in mezzo risospensione riscaldata a 37 ° C e rimuovere le rimanenti parti di non-pancreaticatessuti. Immergere il pancreas a ~ 5 ml di terreno digestione pre-riscaldato in un scintillazione flaconcino da 20 ml. Tritare il pancreas a pezzi di 1-3mm. Mettere in un bagno di acqua riscaldata (in modo ottimale sotto agitazione di ~ 100 rpm). Per accelerare la digestione, pipettare il tessuto digerito alto e verso il basso una volta ogni pochi minuti. NOTA: La durata della collagenasi digestione dipende principalmente dalla concentrazione collagenasi e dalla quantità di forza meccanica applicata sul tessuto. Uso di 0,75 mg / ml di collagenasi per circa 15 min e pipettaggio il tessuto con una pipetta da 5 ml in polistirolo con 2 mm foro di passaggio di ogni 3-5 minuti, con conseguente acini di 10-30 cellule. Dopo la digestione, la sospensione appare torbido, contenente frammenti visibili di tessuto. Dal Collagenasi efficienza varia tra i diversi tipi commerciali e lotti, determinare i tempi di digestione empiricamente. </li><li> Lavaggio e filtraggio <ol><li> Trasferire il tessuto digerito in una provetta da 50 ml e terminare digestion aggiungendo 45 ml di mezzo di risospensione. Filtrare la sospensione attraverso una grossolana (~ 1 mm) rete metallica in una nuova provetta. Centrifugare la flow-through a 100 × g per 3 min. </li><li> Eliminare il surnatante e risospeso il pancreas digeriti in 50 ml di mezzo di risospensione. Filtrare la sospensione attraverso una maglia di nylon 70 o 100 micron. Centrifugare come prima. </li><li> Al fine di rimuovere galleggiante, acini danneggiati, scartare il surnatante e risospendere il tessuto pancreatico in 3 - 5 ml di mezzo di risospensione. Applicare 1 ml di acini pancreatici in 15 ml provette coniche riempiti con 7 ml di terreno risospensione. Lasciare che il acini di stabilirsi per 2 - 3 min. Raccogliere la acini che si sono stabiliti sul fondo della provetta con una pipetta 1 ml, e ripetere questo passaggio 2 - 3 volte. </li><li> Visualizza il acini pancreas al microscopio ottico o dissezione per testare la loro condizione. Nella figura 2, osservare la purezza di isolamento e danni inflitti alle cellule Duriisolamento ng. Utilizzare questi acini immediatamente o cultura per un massimo di 20 ore. </li></ol></li></ol></li></ol> 2 Amilasi secrezione Assay NOTA: Il test secrezione di amilasi serve come misura globale per la capacità secretoria di acini pancreatici. Ciò si ottiene raccogliendo il mezzo in cui gli acini pancreatici sono stati incubati, e determinando l&#39;attività dell&#39;amilasi del mezzo, in generale mediante un kit commerciale. <ol><li> Determinare il numero di condizioni sperimentali da esaminare (ad esempio vs non-stimolata stimolato). Utilizzare almeno tre repliche per ogni condizione. Inoltre, aggiungere un set di repliche per determinare il contenuto totale cellulare amilasi (campione &#39;totale&#39;) e un set per l&#39;attività amilasi del medium (campione di &#39;zero&#39;). Etichettare una piastra multi-pozzetto in cui sarà condotta l&#39;analisi, e due serie di microtubi per ciascuna delle repliche da utilizzare. NOTA: a 12 o 24 pozzettis può essere utilizzato per monitorare 1 e 0,5 ml di sospensioni acinose, rispettivamente. </li><li> Lavare la acini due volte in 30 ml di KRBH media di risospensione e incubare in mezzo risospensione per 30 minuti a 37 ° C. Per la piastra multi-bene aggiungere una goccia 10μl di secretagogue per raggiungere la concentrazione finale appropriato. </li><li> Acini pancreatici rispondono a un gradiente di secretagogo in maniera bi-fasica. Le concentrazioni con valori ottimali di secrezione sono 50 - 100 pM di colecistochinina (CCK) e 1μM di Carbachol (figura 3A) 5. </li><li> Lavare e risospendere il acini in mezzo risospensione. Assicurarsi che le acini sono distribuiti in modo omogeneo nelle sospensioni e aliquote uguali quantità di sospensione nella piastra multi-bene e nei &#39;microtubi total&#39; marcato. Incubare la piastra per 30 minuti a 37 ° C. (Preferibilmente in un bagno di acqua sotto agitazione lieve (~ 50 min)). NOTA: Il numero esatto di acini può variare tra sperits ma dovrebbero essere simili in replicati all&#39;interno di ogni esperimento. Così, la suddivisione in aliquote precisa della sospensione acini è fondamentale per il confronto tra le diverse repliche e condizioni. </li><li> Durante l&#39;incubazione, produrre il &#39;totale&#39; e campioni &quot;zero&quot;. Centrifugare le microprovette &quot;totale&quot; per pochi secondi a 5000 × g e rimuovere 50 microlitri dal surnatante in &#39;microtubi zero&#39;-etichettati e metterli a 4 ° C. Lyse acini nei campioni &quot;totale&quot; con l&#39;aggiunta di Triton X-100 per le microprovette per raggiungere una concentrazione finale di 1%. Agitare vigorosamente le microprovette. NOTA: Aliquote il &#39;totale&#39; replica simile alle repliche dell&#39;esperimento. Lisi delle acini, porta al rilascio del tenore totale di amilasi. In alternativa, raccogliere gli acini in ogni condizione e lisi, alla fine dell&#39;esperimento, per determinare il contenuto cellulare di ogni replica. </li><li> Raccogliere la maggior parte della sospensione acinare into la prima serie di microtubi pre-etichettati. Centrifugare le microprovette per pochi secondi a 5.000 × g. Trasferire il surnatante nella seconda serie dei microtubi pre-etichettati. </li><li> Analizzare il surnatante per l&#39;attività amilasica con un kit commerciale. Calcolare la percentuale di amilasi secreta. NOTA: E &#39;fondamentale leggere l&#39;attività amilasica alla sua gamma lineare. Presentare la percentuale di amilasi secreta come la percentuale di amilasi liberato dal contenuto totale iniziale. Sottrarre il livello di fondo amilasi del mezzo da ogni triplice copia e dividerlo per il contenuto totale iniziale simile normalizzata. </li></ol> 3 Immagini dal vivo della secrezione pancreatica La secrezione da acini pancreatici può essere monitorato direttamente da immagini dal vivo. Utilizzando vari coloranti fluorescenti e sensori, imaging dal vivo permette la caratterizzazione di secrezione ad una risoluzione sub-cellulare. <ol><li> Preparare medio risospensione appena ossigenato come in punto 1.1 e portare a 37 ° C. Impostare il microscopio, e se dotato di un regolatore di temperatura, regolare a 37 ° C. </li><li> Lavare le cellule acinose in 30 ml di terreno di risospensione e metterli in un vetro o di plastica diapositiva. Nota: Se coloranti lipofili devono essere utilizzati per etichettare Per membrana plasmatica etichettati con coloranti lipofili, macchiare la acini per non più di 5 minuti per evitare l&#39;etichettatura delle membrane interne per endocitosi. Durante il lavoro, gestire la acini dolcemente ed evitare un&#39;eccessiva pipettaggio. </li><li> Una volta al microscopio, acini immagine la cui morfologia è chiara, ed evitare l&#39;imaging di acini che visualizzano vesciche basolaterale o vacuoli intracellulari. Aggiungi secretagogue goccia a goccia o utilizzando una camera di perfusione, durante o immediatamente prima dell&#39;inizio di imaging. NOTA: A lenti ad alto ingrandimento con alte aperture numeriche (ad esempio x 63 / 1,4 o × 100 / 1.4) si raccomanda di osservare le strutture subcellulari. </li></ol> 4. O / N Cultura del pancreas Acini (cellulare Alternate Cultura Tecnica) NOTA: O / N coltura viene spesso utilizzato per l&#39;infezione da virus adeno. L&#39;infezione viene condotta a 10 6 -10 7 pfu / ml per 9 - 16 ore prima dell&#39;esame. <ol><li> Cultura acini pancreatici per un massimo di 20 ore. Acini risospese ottenuto da un unico pancreas in 25 ml di terreno di coltura (DMEM supplementato con siero fetale di vitello (FCS) (5%), sodio piruvato (1%), antibiotici (1%), L-glutammina (0,5%), BSA (10 mg / ml) e STI (0,2 mg / ml), e diviso per cinque piastre 10 centimetri. Infect acini con virus in un armadietto livello 3 biosicurezza. </li><li> Incubate acini pancreatici a 37 ° C in aria umidificata 5% di CO 2. Consenti acini di recuperare in media risospensione ossigenato per 30 minuti prima di successivo esame 3,8. </li></ol>

<ol>
	<li>Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pancreatic+acinar+cell+function.">Regulation of pancreatic acinar cell function.</a> Current Opinion in Gastroenterology. 22, 498-504 (2006).</li><li>Schnekenburger, J., et al. <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+kinesin,+dynein+and+microtubules+in+pancreatic+secretion.">The role of kinesin, dynein and microtubules in pancreatic secretion.</a> Cellular and Molecular Life Sciences : CMLS. 66, 2525-2537 (2009).</li><li>Geron, E., Schejter, E. D., Shilo, B. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+exocrine+secretory+vesicles+to+the+apical+membrane+by+actin+cables+generated+by+the+formin+mDia1.">Directing exocrine secretory vesicles to the apical membrane by actin cables generated by the formin mDia1.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 10652-10657 (2013).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characterization+of+isolated+pancreatic+exocrine+cells.">Structural and functional characterization of isolated pancreatic exocrine cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 69, 3028-3032 (1972).</li><li>Williams, J. A., Korc, M., Dormer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+secretagogues+on+a+new+preparation+of+functionally+intact,+isolated+pancreatic+acini.">Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.</a> The American journal of physiology. 235, 517-524 (1978).</li><li>Jerdeva, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+and+non-muscle+myosin+II+facilitate+apical+exocytosis+of+tear+proteins+in+rabbit+lacrimal+acinar+epithelial+cells.">Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells.</a> Journal of Cell Science. 118, 4797-4812 (2005).</li><li>Menozzi, D., Jensen, R. T., Gardner, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Rho+and+Rac+in+secretagogue-induced+amylase+release+by+pancreatic+acini.">A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini.</a> Am J Physiol Cell Physiol. 289, C22-C32 (2005).</li><li>Palade, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+aspects+of+the+process+of+protein+synthesis.">Intracellular aspects of the process of protein synthesis.</a> Science. 189, 867 (1975).</li><li>Masedunskas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+the+actomyosin+complex+in+regulated+exocytosis+revealed+by+intravital+microscopy.">Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 13552-13557 (2011).</li><li>Petersen, H., Grossman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+exocrine+secretion+in+anesthetized+and+conscious+rats.">Pancreatic exocrine secretion in anesthetized and conscious rats.</a> The American Journal of Physiology. 233, E530-E536 (1977).</li><li>Riedl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5, 605-607 (2008).</li><li>Porat-Shliom, N., Milberg, O., Masedunskas, A., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+for+the+actin+cytoskeleton+during+regulated+exocytosis.">Multiple roles for the actin cytoskeleton during regulated exocytosis.</a> Cellular and Molecular Life Sciences : CMLS. 70, 2099-2121 (2013).</li></ol>

The authors declare they have no competing financial interests.

Oltre alla cultura ex vivo, configurazioni alternative per lo studio della secrezione esocrina includono la microscopia intravitale e la misurazione di fluidi dal dotto pancreatico. Microscopia intravitale ha dimostrato di operare bene nel topo ghiandole salivari 10. Questo metodo può registrare la secrezione in tempo reale nel intatta di un animale, ma è limitato nella sua risoluzione e dai mezzi per manipolare il tessuto esocrino. Valutare la secrezione attraverso la raccolta di fluidi dal dotto ...

Il pancreas esocrino costituisce la maggior parte della massa del pancreas mammiferi ed è dedicata alla produzione e secrezione di enzimi digestivi 1. L&#39;unità funzionale del pancreas esocrino, l&#39;acino, è un ammasso di cellule epiteliali che formano e condivide un lume congiunta. Dopo stimolazione ormonale o neuronale, vescicole piene di enzimi vengono trasportati alla superficie della cellula apicale, si fondono con esso ed espellono il loro contenuto nel lume 1-3. Gli enzimi secreti vengono poi svuotati da una serie coalescenza di condotti nel piccolo intestino, dove si catalizzano la ripartizione del cibo in sostanze nutritive. Questo video dimostra come acini intatti sono isolate dal pancreas intero e come la loro capacità secretoria può essere valutata. Utilizzando questa impostazione, la secrezione pancreatica viene quantificato misurando la quantità relativa di amilasi che è stato rilasciato dopo stimolazione. In alternativa, la secrezione può essere realizzata live dall&#39;uso di differentisensori e coloranti. L&#39;ex vivo installazione di acini pancreatici è vantaggiosa in quanto acini isolato, che conservano molte caratteristiche del pancreas intatti, può essere manipolata e controllata più facilmente che in tutta animali 4-6. Questa configurazione è stato originariamente sviluppato alla fine degli anni 1970 ed è stato dato prorogato per lo studio di diversi tessuti esocrine, come salivari e ghiandole mammarie 3-7. In particolare, consente lo studio della secrezione con la minima interferenza alle complesse organizzazioni cellulari di questi epiteli polarizzati.

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ICR mice</td>
			<td style="width:115px;">Harlan</td>
			<td style="width:91px;"></td>
			<td style="width:203px;">Any strain will do</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS</td>
			<td>Sigma Aldrich</td>
			<td>H8264</td>
			<td>Can substitude for KRB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td>Fraction V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor</td>
			<td>Sigma Aldrich</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase XI&#160;</td>
			<td>Sigma Aldrich</td>
			<td>C0130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nylon mesh</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td>70-100&#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amylase infinity reagent&#160;</td>
			<td>Thermo</td>
			<td>TR25421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CCK</td>
			<td>Sigma Aldrich</td>
			<td>C2175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FM4-64</td>
			<td>Lifetechnologies</td>
			<td>F34653</td>
			<td>Diluted to 16&#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20mL scintillation vial</td>
			<td>Sigma Aldrich</td>
			<td>Z190527</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DeltaVision system&#160;</td>
			<td>Applied Precision</td>
			<td></td>
			<td>Consisting of an inverted microscope IX71 equipped with 60x/1.4 or 100x/1.3 objectives (Olympus)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss 510 or 710 confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>60x/1.4 or 100x/1.4 objectives&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Microplate reader</td>
			<td>BioTek</td>
			<td>ELx808</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing the secretory capacity of pancreatic acinar cells

Cellule acinose pancreatiche producono e secernono enzimi digestivi. Queste cellule sono organizzate come un gruppo che forma e condivide un lume congiunta. Questo lavoro dimostra come la capacità secretoria di queste cellule può essere valutata mediante coltura di isolati acini. La configurazione è vantaggiosa in quanto acini isolato, che conservano molte caratteristiche del pancreas esocrino intatto può essere manipolata e controllata più facilmente che in tutta animali. Isolamento corretto di acini pancreas è un requisito chiave in modo che la cultura ex vivo rappresenterà la natura in vivo degli acini. Il protocollo dimostra come isolare acini intatti dal pancreas del mouse. Successivamente, due metodi complementari per la valutazione della secrezione pancreatica sono presentati. Il dosaggio secrezione dell&#39;amilasi serve come misura globale, mentre l&#39;imaging diretto della secrezione pancreatica permette la caratterizzazione della secrezione ad una risoluzione sub-cellulare. Collettivamente, le tecniche Presented qui consentono un ampio spettro di esperimenti per studiare la secrezione esocrina.

Acini pancreas che sono stati isolati correttamente visualizzare una morfologia stereotipata se visti da luce trasmessa. I loro domini basolaterali dovrebbero apparire rotondo e privo di vesciche. Il dominio apicale è circondato da centinaia di vescicole secretorie e appare di colore più scuro (Figura 2A, B). I nuclei sono situati basale alla zona vescicolare. Detriti cellulari e componenti del sistema duttale del pancreas e del pancreas endocrino, che può essere rilevato nelle fasi iniziali di acini...

Watch this Scientific Journal Video about Assessing the Secretory Capacity of Pancreatic Acinar Cells at JoVE.com

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Isolato acini pancreatici mantengono la loro morfologia e l&#39;attività in vivo ed offrono modi potenti per il controllo e la manipolazione della secrezione. Questo lavoro dimostra come acini possono essere isolate dal pancreas del mouse, e come le loro capacità di secrezione possono essere valutati.

Valutare la capacità secretoria delle cellule pancreatiche acinari

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

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JoVE Journal

Biology

16.5K Views.  Weizmann Institute of Science.  Isolato acini pancreatici mantengono la loro morfologia e l'attività in vivo ed offrono modi potenti per il controllo e la manipolazione della secrezione. Questo lavoro dimostra come acini possono essere isolate dal pancreas del mouse, e come le loro capacità di secrezione possono essere valutati. 

Video: Assessing the Secretory Capacity of Pancreatic Acinar Cells

Murine Pancreatic Islet Isolation

Murino pancreatica isolamento Islet

Ricerca

Biologia

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Il trapianto di isole pancreatiche nella capsula renale di topi diabetici

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Isolamento delle isole pancreatiche umane: Parte I: Digestione e Collezione di tessuto pancreatico

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolamento di cellule staminali da xenotrapianti di cancro pancreatico umano

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

Preparazione delle cellule acinose pancreatiche per lo Scopo di Imaging di calcio, cellulari lesioni Misure e Infezione Adenoviral

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Isolamento e Cultura del mouse principale acinose pancreatiche Cellule

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

Fenotipizzazione mouse Pulmonary Function<em&gt; In Vivo</em&gt; Con la capacità polmonare Diffusione

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

Capacità di differenziazione delle cellule progenitrici umane aortica perivascolare adiposo

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imaging calcio Dynamics in sottopopolazioni di cellule di islet pancreatica del topo

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat metabolic syndrome. Thermogenic tissues such as brown and beige fats develop highly specialized mitochondria for heat production. Mitochondria of other tissues, which primarily produce ATP, also convert up to 25% of the total mitochondrial energy production into heat and can, therefore, have a considerable impact on the physiology of the whole body. Mitochondrial thermogenesis is not only essential for maintaining the body temperature, but also prevents diet-induced obesity and reduces the production of reactive oxygen species (ROS) to protect cells from oxidative damage. Since mitochondrial thermogenesis is a key regulator of cellular metabolism, a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction. Importantly, the precise molecular mechanisms that control acute activation of thermogenesis in mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for the direct measurement of uncoupling proteins. The recent development of patch-clamp methodology applied to mitochondria enabled, for the first time, the direct study of the phenomenon at the origin of mitochondrial thermogenesis, H+ leak through the IMM, and the first biophysical characterization of mitochondrial transporters responsible for it, the uncoupling protein 1 (UCP1), specific of brown and beige fats, and the ADP/ATP transporter (AAC) for all other tissues. This unique approach will provide new insights into the mechanisms that control H+ leak and mitochondrial thermogenesis and how they can be targeted to combat metabolic syndrome. This paper describes the patch-clamp methodology applied to mitochondria to study their thermogenic capacity by directly measuring H+ currents through the IMM.

This method article details the main steps in measuring H+ leak across the inner mitochondrial membrane with the patch-clamp technique, a new approach to study the thermogenic capacity of mitochondria.

L'uso della tecnica Patch-Clamp per studiare la capacità termogenica dei mitocondri

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat ...