Wir möchten Renee L. Pasker und Harpreet K. Brar für kompetente technische Unterstützung auf die in dieser Arbeit beschriebenen Protokolle danken. Darüber hinaus möchten wir die Betreuung von Christopher B. Newgard an der Duke University und Alan D. Attie an der Universität von Wisconsin-Madison bestätigen, zusammen mit der Unterstützung ihrer Mitglieder Labor, die uns die Zeit erlaubt und unterstützt notwendig zur Optimierung der beschriebenen Protokollen. Insbesondere danken wir Hans Hohmeier, Danhong Lu, und Helena Winfield in der Labor-und Newgard Mary Rabaglia in der Attie Labor für produktive Diskussionen und Beratung. Diese Arbeit wurde vom NIH DK080845 und Juvenile Diabetes 594 unterstützt Research Foundation Zuschuss 17-2011-608 (MEK)

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Autoren haben nichts zu offenbaren.

Mit der Verbreitung von Diabetes voraussichtlich um 7,7% der Weltbevölkerung betreffen, ist die Voraussetzung für neuartige Forschungstechniken unerlässlich, sowohl Diabetes verstehen und zu behandeln 18. Die vorliegende Inselisolierung ist ein gut etabliertes Protokoll für die in-vitro-Versuche verwendet und hat vorher mit leichten Modifikationen 11,14,16 vorgestellt. Obwohl die Insulinsekretion ist ein gemeinsames stromabwärts Anwendung isolierten Inseln, die sich auf stromaufwärts...

Die strikte Einhaltung euglycemia ist zwingend notwendig, um Begleiterkrankungen wie Neuropathie, Nephropathie, Retinopathie und, die alle Markenzeichen der Pathologie des unkontrollierten Typ-1-und-2-Diabetes sind 1 zu verhindern. Reduzierte β-Zell-Funktion und Masse in Typ 1 und 2 Diabetes stören Blutglukosekonzentrationen 2. Während immun-vermittelte Typ-1-Diabetes resultiert aus einem verheerenden Verlust von Insulin-produzierenden β-Zellen, gestörte β-Zellen Insulin-Sekretion und periphere Insulin-Signal bei Typ-2-Diabetes zusammen fördern Hyperglykämie, Dyslipidämie und erhöhte hepatische Glukoseproduktion, die schließlich zu sowohl Verlust der β-Zellmasse und Insulin sekretorischen Kapazität von einzelnen β-Zellen 3. Das Verständnis der zugrunde liegenden Mechanismen β-Zellen in der Progression des Typ-1-und-2-Diabetes wird hoffentlich Anlass zu neuen Therapien zur Vorbeugung und Behandlung dieser Krankheiten. In-vitro-tiUSGABE Kulturmodelle, wie der INS-1 und β-MIN6 verewigt Zelllinien können nützliche Werkzeuge für das Verständnis spezifischer β-Zell-Funktionen sein. Jedoch sind die Wechselwirkungen zwischen den verschiedenen Zelltypen innerhalb der Insel können sich regulieren β-Zellfunktion. Beispielsweise die parakrine Einfluss von Glucagon (aus α-Zellen freigesetzt) ​​und Somatostatin (von δ-Zellen freigesetzt) ​​in zunehmenden und abnehm Insulinsekretion bzw. zeigt die Bedeutung der Zell Zelle in der Nähe des endokrinen Reaktion 4. Darüber hinaus gap junctions zwischen β-Zellen verstärken die Freisetzung von Insulin 5. Obwohl Fortschritte wurden bei der Erzeugung Insulinom Leitungen, die besser repliziert die physiologische Reaktion von isolierten Inseln zu Glucose vorliegen (z. B. das INS-1-abgeleiteten 832/13 und 832/3 Zelllinien), die Glucose-Empfindlichkeit immer noch von normalen Ratten unterscheidet Inselchen 6,7. Darüber hinaus ist die Antwort dieser klonalen Zellinien InsulinomGlucagon-like-Peptid-1 (GLP-1)-Agonisten kann drastisch voneinander aus normalen Inseln 6 unterscheiden, sowie. Daher kann immortalisierte Zelllinien das beste Modell für die Untersuchung Agenten, die Auswirkungen auf die cAMP-Produktion nicht zu vertreten. Im Gegensatz zu den Insulinom-Zelllinien, studiert β-Zellfunktion nur in Ganztiermodellen besitzt einen eigenen Satz von Komplikationen. Eine der größten Herausforderungen in der Arbeit mit endokrinen Gewebe präzise Messung der Konzentration des Hormons freigesetzt. Insbesondere spielt die Leber eine wichtige Rolle metabolisierenden Insulin und die Bauchspeicheldrüse Blutfluss geht direkt in die Leber. Somit kann eine Plasmainsulinmessung nicht genau schildern die Mengen an Insulin aus der Bauchspeicheldrüse oder der Auswirkung von verschiedenen Behandlungen auf die Geschwindigkeit der Insulinabsonderung 8 sezerniert. Darüber hinaus kann Nierenstoffwechsel von Glucagon die Zuverlässigkeit von Glucagon Ausgabe von α-Inselzellen 9 begrenzen. Daher isolieren primären Maus Inseln für in-vitro-Experimenten eine genauere Verständnis davon, wie die Insel ist auf bestimmte Reize reagieren zu ergänzen Messungen in vivo gemacht. Das vorliegende Protokoll für die Isolierung von Maus-Inseln ist ein gut etabliertes Protokoll, das von einer Reihe von Gruppen (mit leichten Modifikationen, die helfen, den Erfolg zu erhöhen) 10,11 eingesetzt. Darüber hinaus ist die Bestimmung der cAMP-Produktion erlaubt eine direkte Auslesen des Inkretin Ansprechbarkeit der β-Zellen. In Verbindung mit cAMP-Messung, Proteingehalt und Insulinsekretion kann auch aus dem gleichen Lager Probenvorbereitungs quantifiziert werden, zu helfen, um zu bestimmen, ob ein Defekt in der β-Zell-Funktion liegt proximal oder distal zu cAMP 10. Die endgültige cAMP-Gehalt und die Insulinsekretion Anwendung in diesem Protokoll kann ein sehr mächtiges Werkzeug für das Verständnis der Einfluss der Pharma-und Nahrungsbestandteile, unter anderen seins, auf die cAMP-und Insulinsekretion. Neben der Stimulation von Glukose allein, können auch andere Verbindungen verwendet werden, um Veränderungen in der cAMP-und Insulin-Sekretion 10,11 messen. Schließlich, obwohl Insulin ist die primäre Hormon wir Assay von isolierten Inseln, andere Hormone, wie Glucagon und Somatostatin, sowie Zytokine, Eicosanoide und cyclischem Adenosinmonophosphat, kann ebenfalls gemessen werden, entweder durch eine vorübergehende Stimulation Assay oder durch Quantifizierung ihre Niveaus in Kulturmedium 12. Schließlich, obwohl außerhalb des Geltungsbereichs dieses Manuskripts Inselisolierung mit der beschriebenen Kollagenase Isolationsmethode ermöglicht Insel Erhaltung so, dass viele andere Downstream-Anwendungen verfolgt werden, wie zum Beispiel Inseltransplantation, RNA-Isolierung für die quantitative Echtzeit-PCR-oder Microarray-Analysen, Protein Isolation für Western Blot, Immunfluoreszenz-und Insel Einbettung Bildgebung und [3H]-Thymidin als Maß für die Inselzell-ReplikationKation, von denen einige in früheren JoVE Artikel 13-16 beschrieben. Insgesamt nach der im Protokoll beschriebenen Inselisolierung Verfahren kann ein Forscher mit wichtigen und nützlichen Informationen für die Entwicklung von Therapien und Förderung der Arzneimittelforschung zur Verbesserung β-Zell-Funktion ausgerichtet ist.

<table border="0" cellpadding="0" cellspacing="0" width="502">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Collagenase: Collagenase from Clostridium histolyticum suitable for isolating active islets</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C7657</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Ficoll 400</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>F9378</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hanks Balanced Salt Solution 10X</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td>14065-056</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hepes</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>H3375</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">RPMI 1640 (powder)</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">31800-022</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>A7888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3/0 Silk Suture Thread</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>18020-30</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dumont #5 Forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>11251-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">0.8 mm Forceps&#160;&#160;</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">11050-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Curved Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14061-10</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">15003-08</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dissecting Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14002-14</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">5ml BD Luer-Lok Syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309646</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">1ml BD syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309628</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">30 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305106</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">27 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305109</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sharpening Stone</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">29008-01</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-2-2 tribromoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">T48402-25G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-methyl-2-butanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>240486-100mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sodium Chloride (NaCl)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Potassium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>P3911</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:235px;">Monopotassium Phosphate (KH2PO4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0662</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Sodium Bicarbonate (NaCHO3)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">S6014</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl2 *2H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C3881</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO4 *7H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">M9397</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">15140-122</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:235px;">Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:136px;">SH30088.03HI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">5879-100MG</td>
		</tr>
	</tbody>
</table>

a method for mouse pancreatic islet isolation and intracellular camp determination

Unkontrollierter Blutzucker ist ein Markenzeichen von Diabetes mellitus und Begleiterkrankungen fördert wie Neuropathie, Nephropathie und Retinopathie. Mit der zunehmenden Verbreitung von Diabetes, sowohl immun-vermittelte Typ-1-und Adipositas-Linked-Typ-2-Studien bei der Abgrenzung Diabetes Pathophysiologie und therapeutische Mechanismen abzielen, sind von entscheidender Bedeutung. Die β-Zellen der Langerhans-Inseln Langerhans sind entsprechend Absonderung von Insulin in Reaktion auf einen erhöhten Blutzuckerspiegel verantwortlich. Zusätzlich zu Glukose und anderen Nährstoffen sind die β-Zellen auch durch spezifische Hormone stimuliert, Inkretine bezeichnet, die aus dem Darm in Reaktion auf eine Mahlzeit und wirken auf β-Zellen-Rezeptoren, die die Produktion von intrazellulären zyklischen Adenosinmonophosphat zu erhöhen sezerniert werden ( cAMP). Verringerte β-Zell-Funktion, Masse und Inkretin Reaktionsfähigkeit sind gut zu verstehen, die das Entstehen der Typ-2-Diabetes beitragen, und werden auch zunehmend verknüpft with Typ 1 Diabetes. Die vorliegende Maus Inselisolierung und cAMP-Bestimmung Protokoll kann ein Werkzeug, um zu beschreiben Mechanismen zur Förderung Fortschreiten der Krankheit und Therapiemaßnahmen, insbesondere solche, die von den Inkretin-Rezeptoren vermittelt werden oder verwandte Rezeptoren, die durch Modulation der intrazellulären cAMP-Produktion handeln. Während nur cAMP-Messungen beschrieben werden wird, schafft das beschriebene Inselisolierung Protokoll eine saubere Präparation, ermöglicht auch viele andere Downstream-Anwendungen, einschließlich Glucose-stimulierten Insulinsekretion, [3 H]-Thymidin-Einbau, Proteinmenge und der mRNA-Expression.

Um eine hohe Ausbeute bei der Isolierung Insel zu gewährleisten, sollten chirurgische Techniken im Protokoll beschriebenen genau befolgt werden. Obwohl die hier vorgestellten Verfahren wird jedem Labor zugeschnitten werden, gibt es ein paar wichtige Schritte, die zu einem erfolgreichen Isolierung führen. Um den gemeinsamen Gallengang leicht zugänglich zu machen, ist es empfehlenswert, dass die Organe auf der rechten Seite der Maus (Fig. 1) verschoben werden kann. Darüber hinaus ermöglicht dies die ...

Watch this Scientific Journal Video about A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination at JoVE.com

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Die Untersuchung in vitro β-Zell-Funktion mit isolierten Maus-Langerhans-Inseln ist ein wichtiger Bestandteil in der Studie der Diabetes Pathophysiologie und Therapie. Während viele Downstream-Anwendungen zur Verfügung stehen, dieses Protokoll beschreibt speziell die Messung der intrazellulären zyklischen Adenosinmonophosphat (cAMP) als wesentlicher Parameter, der β-Zellfunktion.

Eine Methode zur Maus-Inselzellen der Bauchspeicheldrüse Isolation und intrazellulären cAMP-Bestimmung

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

a-method-for-mouse-pancreatic-islet-isolation-intracellular-camp

Research

JoVE Journal

Biology

65.1K Aufrufe.  University of Wisconsin-Madison.  Die Untersuchung in vitro β-Zell-Funktion mit isolierten Maus-Langerhans-Inseln ist ein wichtiger Bestandteil in der Studie der Diabetes Pathophysiologie und Therapie. Während viele Downstream-Anwendungen zur Verfügung stehen, dieses Protokoll beschreibt speziell die Messung der intrazellulären zyklischen Adenosinmonophosphat (cAMP) als wesentlicher Parameter, der β-Zellfunktion. 

Serie: A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Murine Pancreatic Islet Isolation

Murine Inselzellen der Bauchspeicheldrüse Isolation

Forschung

Biologie

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.

Menschliche Inselzellen der Bauchspeicheldrüse Isolation: Part I: Verdauung und Sammlung von Pankreasgewebe

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

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

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 characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we 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. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

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 II: purification and culture of human islets) using a modified automated method.

Menschliche Inselzellen der Bauchspeicheldrüse Isolation: Part II: Reinigung und Kultur der Menschenrechte Islets

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

Computer-assisted Großflächige Visualisierung und Quantifizierung der Inselzellen der Bauchspeicheldrüse Masse, Größenverteilung und Architektur

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Maus Islet der Langerhans Isolation durch eine Kombination von gereinigtem Collagenase und Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

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.

Isolierung und Kultivierung von primären Maus Azinuszellen des Pankreas

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 ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

Nahes Infrarot (NIR) Licht Erhöht Ausdruck einer Markierung der Funktion der Mitochondrien im Maus Vestibular Sinnesepithel

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Ein einfaches Hocheffizienzprotokoll für die Isolierung von Pankreas-Isleten von Mäusen

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Eine Methode zur Inselversetzung, Omentum in Maus

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Pancreatic Islet Einbettung für Paraffin Abschnitte

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...