Nous tenons à remercier Renée L. Pasker et Harpreet K. Brar pour l&#39;assistance technique d&#39;experts sur les protocoles décrits dans ce travail. En outre, nous tenons à remercier le mentorat de Christopher B. Newgard à l&#39;Université Duke et Alan D. Attie à l&#39;Université de Wisconsin-Madison, avec le soutien des membres de leur laboratoire, qui nous ont permis le temps et le soutien nécessaires pour optimiser la protocoles décrits. En particulier, nous remercions Hans Hohmeier, Danhong Lu, et Helena Winfield dans le laboratoire Newgard et Marie Rabaglia dans le laboratoire Attie pour des discussions et des conseils productifs. Ce travail a été soutenu par le NIH DK080845 de subvention et le diabète juvénile 594 Fondation de la recherche subvention 17-2011-608 (MEK)

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Les auteurs ont rien à révéler.

Avec la prévalence du diabète devrait affecter 7,7% de la population mondiale, l&#39;exigence de nouvelles techniques de recherche est impératif à la fois comprendre et traiter le diabète 18. L&#39;isolement des îlots présente est un protocole bien établi utilisés pour l&#39;expérimentation in vitro et a été présenté précédemment, avec de légères modifications 11,14,16. Bien que la sécrétion d&#39;insuline est une application en aval commun pour des îlots isolés, en s...

Le maintien strict de euglycémie est impératif de prévenir les pathologies telles que la neuropathie, la néphropathie et la rétinopathie, qui sont toutes des caractéristiques de la pathologie de incontrôlée de type 1 et 2 de diabète 1. Fonction β-cellulaire réduite et la masse à la fois de type 1 et diabète de type 2 perturbent les concentrations de glucose dans le sang 2. Alors que le type auto-immune 1 diabète résulte d&#39;une perte dévastatrice de cellules β productrices d&#39;insuline, troubles de β-cellulaire sécrétion d&#39;insuline et périphérique signalisation de l&#39;insuline dans le diabète de type 2, ainsi que de promouvoir l&#39;hyperglycémie, la dyslipidémie, et augmentation de la production hépatique de glucose, ce qui entraîne par la suite dans à la fois la perte de masse β-cellulaire et de l&#39;insuline sécrétoire capacité de β-cellules individuelles 3. Comprendre les mécanismes sous-jacents β-cellules dans la progression de type 1 et diabète de type 2, nous l&#39;espérons donner lieu à de nouvelles thérapies pour prévenir et traiter ces maladies. Dans ti vitrodes modèles de culture uestion, tels que l&#39;INS-1 et MIN6 immortalisés lignes β-cellulaires, peuvent être des outils utiles pour la compréhension des fonctions spécifiques β-cellules. Cependant, les interactions entre les différents types de cellules au sein de l&#39;îlot peut se réguler la fonction β-cellulaire. Par exemple, l&#39;influence paracrine de glucagon (libérée à partir de cellules-α) et la somatostatine (libéré à partir des cellules-δ) en augmentant et en diminuant la sécrétion d&#39;insuline, respectivement, démontre l&#39;importance de la proximité de la cellule de la cellule dans la réponse endocrinienne 4. En outre, les jonctions communicantes entre les cellules-β potentialisent la libération d&#39;insuline 5. En outre, bien que des progrès ont été réalisés dans la génération de lignées insulinome que mieux reproduits la réponse physiologique des îlots isolés au glucose (par exemple, l&#39;INS-1 dérivé 832/13 et 832/3 lignées cellulaires), leur réactivité de glucose diffère encore de rat normal îlots 6,7. En outre, la réponse de ces lignées cellulaires d&#39;insulinome clonalesde peptide-1 (GLP-1) des agonistes du glucagon-like peuvent différer considérablement les uns des autres, ainsi que de six îlots normaux. Par conséquent, les lignées cellulaires immortalisées peuvent ne pas représenter le meilleur modèle pour les agents de dosage ayant un impact sur la production d&#39;AMPc. Par contraste avec les lignées cellulaires dérivées insulinome, l&#39;étude de la fonction β-cellulaire uniquement dans des modèles animaux entiers offre sa propre série de complications. Un des plus grands défis dans le travail avec le tissu endocrinien est de mesurer la concentration précise de l&#39;hormone libérée. Plus précisément, le foie joue un rôle majeur métaboliser l&#39;insuline, et le flux sanguin du pancréas va directement vers le foie. Ainsi, une mesure de l&#39;insuline dans le plasma peut ne pas représenter avec précision les quantités d&#39;insuline est sécrétée par le pancréas lui-même ou de l&#39;incidence des différents traitements sur le taux de sécrétion de l&#39;insuline 8. En outre, le métabolisme rénal de glucagon peut limiter la fiabilité de la production de glucagon par α-cellules d&#39;îlots 9. Par conséquent, l&#39;isolement d&#39;îlots de souris primaires pour l&#39;expérimentation in vitro fournit une compréhension plus précise de la façon dont l&#39;îlot est de répondre à des stimuli spécifiques pour compléter les mesures effectuées in vivo. Le présent protocole pour l&#39;isolement des îlots de souris est un protocole bien établi utilisé par un certain nombre de groupes (avec de légères modifications qui peuvent aider à accroître la réussite) 10,11. En outre, la détermination de la production d&#39;AMPc permet une lecture directe de la capacité de réponse des cellules incrétine-β. En conjonction avec la mesure de l&#39;AMPc, la teneur en protéines et la sécrétion d&#39;insuline peut également être quantifié à partir de la même préparation de l&#39;échantillon de l&#39;AMPc, aidant à déterminer si un défaut de la fonction β-cellule se trouve proximal ou distal par rapport à l&#39;AMPc 10. La teneur en AMPc finale et l&#39;insuline demande de sécrétion dans ce protocole peut être un outil très puissant pour comprendre l&#39;influence des constituants pharmaceutiques et alimentaires, entre autres,s, sur l&#39;AMPc et la sécrétion d&#39;insuline. En plus de la stimulation de glucose seul, d&#39;autres composés peuvent être utilisés pour mesurer les changements dans l&#39;AMPc et la sécrétion d&#39;insuline 10,11. Enfin, bien que l&#39;insuline est l&#39;hormone principale nous dosage à partir de d&#39;îlots isolés, d&#39;autres hormones, telles que le glucagon et la somatostatine, ainsi que des cytokines, des eicosanoïdes, et l&#39;adénosine monophosphate cyclique, peut également être mesuré, soit par un test de stimulation transitoire ou par quantification de leurs niveaux dans un milieu de culture 12. Enfin, bien que hors de la portée de ce manuscrit, l&#39;isolement d&#39;îlots avec le procédé d&#39;isolement de la collagénase décrit permet une préservation des îlots de sorte que de nombreuses autres applications en aval peuvent être poursuivis, tels que la greffe d&#39;îlots, l&#39;isolement de l&#39;ARN pour le temps réel de PCR quantitative ou microréseau analyses, protéine l&#39;isolement de transfert de Western, îlot plongement et l&#39;imagerie par immunofluorescence, et l&#39;incorporation de [3H]-thymidine en tant que mesure de repli des cellules des îlotscation, dont certains ont été décrits dans les articles de JoVE précédentes 13 à 16. Dans l&#39;ensemble, la suite de la procédure d&#39;isolement des îlots décrite dans le protocole peut fournir un chercheur de l&#39;information importante et utile pour le développement de thérapies et de promouvoir la découverte de médicaments visant à améliorer la fonction β-cellulaire.

<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

Glycémie non contrôlée est une caractéristique du diabète sucré et favorise morbidités comme la neuropathie, la néphropathie et la rétinopathie. Avec la prévalence croissante du diabète, à la fois auto-immune de type 1 et lié à l&#39;obésité de type 2, des études visant à délimiter le diabète physiopathologie et les mécanismes thérapeutiques sont d&#39;une importance critique. Les β-cellules d&#39;îlots pancréatiques de Langerhans sont responsables de la sécrétion de l&#39;insuline de façon appropriée en réponse à des concentrations élevées de glucose dans le sang. En plus du glucose et d&#39;autres nutriments, les cellules β sont également stimulés par des hormones spécifiques, appelées incrétines, qui sont sécrétées par l&#39;intestin en réponse à un repas et d&#39;agir sur les récepteurs β-cellulaires qui augmentent la production d&#39;adénosine monophosphate cyclique intracellulaire ( cAMP). Diminution de la fonction β-cellulaire, la masse, et la réactivité de l&#39;incrétine sont bien comprises de contribuer à la physiopathologie du diabète de type 2, et sont de plus en plus liés wile diabète de type 1 e. L&#39;isolement actuel de l&#39;îlot de la souris et de l&#39;AMPc protocole de détermination peut être un outil pour aider à délimiter les mécanismes favorisant la progression de la maladie et des interventions thérapeutiques, en particulier ceux qui sont médiés par les récepteurs de l&#39;incrétine ou récepteurs qui agissent par modulation de la production d&#39;AMPc intracellulaire liée. Bien que seules des mesures d&#39;AMPc vont être décrits, le protocole d&#39;isolement des îlots de Langerhans décrit crée une préparation propre, qui permet également de nombreuses autres applications en aval, y compris le glucose stimulé la sécrétion d&#39;insuline, la [3 H]-thymidine, abondance des protéines, et l&#39;expression de l&#39;ARNm.

Pour assurer un rendement élevé au cours de l&#39;isolement des îlots, des techniques chirurgicales décrites dans le protocole devraient être suivis de près. Bien que les techniques présentées ici seront adaptés à chaque laboratoire, il ya quelques étapes essentielles qui conduiront à un isolement réussi. Afin de rendre le canal cholédoque facilement accessible, il est recommandé que les organes soient déplacés vers le côté droit de la souris (Figure 1). En outre, cela permettra le pa...

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

Le dosage in vitro la fonction β-cellulaire en utilisant des îlots isolés de souris de Langerhans est un élément important dans l&#39;étude du diabète physiopathologie et la thérapeutique. Alors que de nombreuses applications en aval sont disponibles, ce protocole décrit précisément la mesure intracellulaire d&#39;adénosine monophosphate cyclique (AMPc) comme un paramètre essentiel de déterminer la fonction β-cellulaire.

Une méthode pour la souris Isolement îlots pancréatiques et AMPc intracellulaire Détermination

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

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64.9K vues.  University of Wisconsin-Madison.  Le dosage in vitro la fonction β-cellulaire en utilisant des îlots isolés de souris de Langerhans est un élément important dans l'étude du diabète physiopathologie et la thérapeutique. Alors que de nombreuses applications en aval sont disponibles, ce protocole décrit précisément la mesure intracellulaire d'adénosine monophosphate cyclique (AMPc) comme un paramètre essentiel de déterminer la fonction β-cellulaire. 

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

Murine Pancreatic Islet Isolation

Isolement murin îlots pancréatiques

Recherche

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.

Isolement îlots pancréatiques humaines: Partie I: La digestion et la perception des tissus pancréatiques

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.

Isolement îlots pancréatiques humaines: Partie II: Purification et culture d&#39;îlots humains

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.

Assistée par ordinateur à grande échelle de visualisation et quantification de la messe des îlots pancréatiques, distribution de taille et d&#39;architecture

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.

Souris îlot de Langerhans isolement à l&#39;aide d&#39;une combinaison de la collagénase purifiée et protéase neutre

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.

Isolement et culture de cellules primaires de souris acineuses du pancréas

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.

Proche infrarouge (NIR) Lumière augmente l&#39;expression d&#39;un marqueur de mitochondrial Fonction chez la souris vestibulaire sensoriel épithélium

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

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Filtration Isolement des acides nucléiques: Une méthode d&#39;extraction de l&#39;ADN simple et rapide

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

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.