Gostaríamos de agradecer a Renee L. Pasker e Harpreet K. Brar para assistência técnica especializada sobre os protocolos descritos neste trabalho. Além disso, gostaríamos de reconhecer a orientação de Christopher B. Newgard na Duke University e Alan D. Attie na Universidade de Wisconsin-Madison, juntamente com o apoio de seus membros de laboratório, o que nos permitiu a tempo e apoio necessários para otimizar o protocolos descritos. Em particular, agradecemos Hans Hohmeier, Danhong Lu, e Helena Winfield no Laboratório Newgard e Maria Rabaglia no Laboratório Attie para discussões produtivas e conselhos. Este trabalho foi financiado pelo NIH DK080845 concessão e diabetes juvenil 594 Fundação de Pesquisa de concessão 17-2011-608 (a MEK)

<ol>
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Com a prevalência de diabetes projetados para afetar 7,7% da população do mundo, a exigência de novas técnicas de pesquisa é fundamental para entender e tratar a diabetes 18. O presente isolamento de ilhotas é um protocolo bem estabelecido utilizado para experimentação in vitro e tem sido apresentado anteriormente, com ligeiras modificações 11,14,16. Embora a secreção de insulina é uma aplicação comum para a jusante ilhotas isoladas, com foco em componentes a montante, tais...

A manutenção rigorosa de euglicemia é imperativo para evitar morbidades, tais como neuropatia, nefropatia e retinopatia, que são todas as características da patologia da descontrolada tipo 1 e 2 diabetes 1. Função das células-β e de massa em ambos tipo 1 e diabetes 2 perturbar as concentrações de glicose no sangue 2. Considerando tipo imune mediada por uma diabetes resulta de uma perda devastadora de células-β produtoras de insulina, a secreção de insulina das células β prejudicada e sinalização da insulina periférica na diabetes tipo 2 em conjunto promover hiperglicemia, dislipidemia e aumento da produção hepática de glicose, o que acaba por resultar em tanto a perda da capacidade de destruição de células β e secreção de insulina a partir de células individuais β-3. Compreender os mecanismos de células-β subjacentes a progressão da diabetes tipo 1 e 2, esperamos dar origem a novas terapias para prevenir e tratar estas doenças. In vitro timodelos de cultura ssue, tais como o INS-1 e MIN6 linhas de células imortalizadas β, podem ser ferramentas úteis para a compreensão da função das células β específicos. No entanto, as interações entre os diferentes tipos de células dentro da ilhota-se pode regular a função das células β. Por exemplo, a influência parácrina de glucagon (libertado a partir de células-α) e somatostatina (libertado a partir de células-δ) em aumentar e diminuir a secreção de insulina, respectivamente, demonstra a importância da proximidade de células de células na resposta endócrina 4. Além disso, junções entre as células-β potencializar a liberação de insulina 5. Além disso, embora tenham sido feitos progressos na geração de linhas de insulinoma que melhor replicados a resposta fisiológica de ilhéus isolados de glicose (por exemplo, o INS-1 derivado de 832/13 e 832/3 linhas de células), a sua capacidade de resposta de glucose ainda difere do normal de rato ilhotas 6,7. Além disso, a resposta destas linhas celulares de insulinoma clonaisa peptide-1 (GLP-1) agonistas de glucagon-like pode variar drasticamente de uma outra, bem como a partir de ilhéus normais 6. Portanto, as linhas de células imortalizadas podem não representar o melhor modelo para os agentes de ensaio que o impacto sobre a produção de cAMP. Em contraste com as linhas de células derivadas de insulinoma, estudar a função das células β exclusivamente em modelos animais completos oferece o seu próprio conjunto de complicações. Um dos maiores desafios no trabalho com tecido endócrino está medindo a concentração precisa de hormônio liberado. Especificamente, o fígado desempenha um papel importante metabolizar a insulina, e o fluxo de sangue pâncreas vai directamente para o fígado. Assim, uma medição de insulina no plasma podem não descrever com precisão a quantidade de insulina a ser segregadas a partir da própria pâncreas ou o impacto de diferentes tratamentos sobre a taxa de secreção de insulina 8. Além disso, o metabolismo renal do glucagon pode limitar a confiabilidade da produção de glucagon a partir de células dos ilhéus α-9. Portanto, isolar ilhotas principal do mouse para a experimentação in vitro proporciona uma compreensão mais precisa de como a ilhota está respondendo a estímulos específicos para complementar as medições feitas in vivo. O presente protocolo para o isolamento de ilhotas de rato é um protocolo bem estabelecido usado por um número de grupos (com pequenas modificações que podem ajudar a aumentar o sucesso) 10,11. Além disso, a determinação da produção de cAMP permite uma leitura directa da capacidade de resposta das células incretina-β. Em conjunção com a medição de cAMP, teor de proteína e a secreção de insulina pode também ser quantificada a partir da mesma preparação de amostras de cAMP, ajudando a determinar se um defeito na função das células-β reside proximal ou distal em AMPc 10. O conteúdo cAMP final e aplicação da secreção de insulina neste protocolo pode ser uma ferramenta muito poderosa para a compreensão da influência de componentes farmacêuticos e dietéticos, entre outross, em cAMP e secreção de insulina. Além disso a estimulação da glucose sozinha, outros compostos podem ser utilizados para medir as alterações do cAMP e secreção de insulina 10,11. Finalmente, embora a insulina é a hormona primária que ensaiar a partir de ilhotas isoladas, outras hormonas, como o glucagon e somatostatina, assim como citoquinas, e eicosanóides, o monofosfato cíclico de adenosina, pode também ser medida, quer por um ensaio de estimulação transitória ou por quantificação de os seus níveis no meio de cultura 12. Finalmente, embora fora do âmbito deste manuscrito, isolamento de ilhotas com o método de isolamento da colagenase descrita permite a preservação da ilhota de forma que muitas outras aplicações a jusante, pode ser exercida, tal como o transplante de ilhotas, para isolamento de RNA por PCR quantitativo em tempo real ou de microarray análises de proteínas isolamento por Western blot, dos ilhéus incorporação e imagiologia de imunofluorescência, e incorporação de [3H]-timidina como medida de repli antiilhotascação, alguns dos quais têm sido descritas em artigos anteriores JoVE 13-16. No geral, seguindo o procedimento de isolamento das ilhotas descritos no protocolo pode fornecer um pesquisador com informações importantes e úteis para o desenvolvimento de terapias e promover a descoberta de medicamentos destinado a melhorar a função das células β.

<table><tbody><tr><td>Collagenase from &lt;em&gt;Clostridium histolyticum&lt;/em&gt; suitable for isolating active islets</td><td>Sigma-Aldrich</td><td>C7657</td><td></td></tr><tr><td>Ficoll 400</td><td>Sigma-Aldrich</td><td>F9378</td><td></td></tr><tr><td>Hanks Balanced Salt Solution 10X</td><td>Invitrogen (Gibco)</td><td>14065-056</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>RPMI 1640 (powder)</td><td>Invitrogen (Gibco)</td><td>31800-022</td><td></td></tr><tr><td>Albumin from Bovine Serum (BSA)</td><td>Sigma-Aldrich</td><td>A7888</td><td></td></tr><tr><td>3/0 Silk Suture Thread</td><td>Fine Science Tools</td><td>18020-30</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>0.8 mm Forceps&amp;nbsp;&amp;nbsp;</td><td>Fine Science Tools</td><td>11050-10</td><td></td></tr><tr><td>Curved Scissors</td><td>Fine Science Tools</td><td>14061-10</td><td></td></tr><tr><td>Vannas-T&amp;uuml;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Dissecting Scissors</td><td>Fine Science Tools</td><td>14002-14</td><td></td></tr><tr><td>5 ml BD Luer-Lok Syringe</td><td>BD</td><td>309646</td><td></td></tr><tr><td>1 ml BD syringe</td><td>BD</td><td>309628</td><td></td></tr><tr><td>30 G BD Needle 1/2&quot; Length</td><td>BD</td><td>305106</td><td></td></tr><tr><td>27 G BD Needle 1/2&quot; Length</td><td>BD</td><td>305109</td><td></td></tr><tr><td>Sharpening Stone</td><td>Fine Science Tools</td><td>29008-01</td><td></td></tr><tr><td>2-2-2-tribromoethanol</td><td>Sigma-Aldrich</td><td>T48402-25G</td><td></td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma-Aldrich</td><td>240486-100mL</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Monopotassium Phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P0662</td><td></td></tr><tr><td>Sodium Bicarbonate (NaCHO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C3881</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M9397</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen (Gibco)</td><td>15140-122</td><td></td></tr><tr><td>Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td><td>Fisher Scientific</td><td>SH30088.03HI</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine (IBMX)</td><td>Sigma-Aldrich</td><td>5879-100MG</td><td></td></tr></tbody></table>

a method for mouse pancreatic islet isolation and intracellular camp determination

Glicemia descontrolada é uma característica da diabetes mellitus e promove morbidades como neuropatia, nefropatia e retinopatia. Com o aumento da prevalência de diabetes, tanto imunomediada tipo 1 e ligado à obesidade do tipo 2, estudos que visam delinear fisiopatologia diabetes e mecanismos terapêuticos são de importância crítica. As células-β dos ilhéus pancreáticos de Langerhans são responsáveis ​​pela apropriadamente secretoras de insulina, em resposta a concentrações elevadas de glicose no sangue. Em adição à glucose e outros nutrientes, as células-β também são estimulados por hormonas específicas, denominado incretinas, que são secretadas a partir do intestino em resposta a uma refeição e agem em receptores de células β que aumentam a produção intracelular de monofosfato de adenosina cíclico ( cAMP). Diminuição da função das células-β, massa e capacidade de resposta incretina são bem compreendidos a contribuir para a fisiopatologia do diabetes tipo 2, e também estão sendo cada vez mais ligada wiª diabetes tipo 1. O presente rato isolamento ilhéu e protocolo determinação cAMP pode ser uma ferramenta para ajudar a delinear os mecanismos que promovem a progressão da doença e intervenções terapêuticas, particularmente aquelas que são mediadas pelos receptores de incretinas ou receptores que atuam através da modulação da produção de cAMP intracelular relacionados. Embora apenas as medições de cAMP será descrito, o protocolo de isolamento de ilhotas descrito cria uma preparação limpa, que permite também a muitas outras aplicações a jusante, incluindo a glicose estimulou a secreção de insulina, [3 H]-timidina, a abundância de proteínas, e a expressão do mRNA.

Para garantir um alto rendimento ilhota durante o isolamento, as técnicas cirúrgicas descritas no protocolo deve ser seguido de perto. Embora as técnicas apresentadas aqui vai ser adaptado para cada laboratório, existem alguns passos críticos que levarão a um isolamento bem sucedido. A fim de tornar o canal biliar comum de fácil acesso, recomenda-se que os órgãos de ser deslocado para o lado direito do rato (Figura 1). Além disso, isto irá permitir que o pâncreas são accionados com uma quan...

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

Ensaiando in vitro a função das células β usando o mouse ilhotas isoladas de Langerhans é um componente importante no estudo da fisiopatologia do diabetes e terapêutica. Embora muitas aplicações a jusante estão disponíveis, este protocolo descreve especificamente a medida da intracelular monofosfato cíclico de adenosina (AMPc) como um parâmetro essencial para definir a função das células β.

Um Método para Mouse ilhotas pancreáticas Isolamento e intracelular cAMP Determinação

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

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65.1K visualizações.  University of Wisconsin-Madison.  Ensaiando in vitro a função das células β usando o mouse ilhotas isoladas de Langerhans é um componente importante no estudo da fisiopatologia do diabetes e terapêutica. Embora muitas aplicações a jusante estão disponíveis, este protocolo descreve especificamente a medida da intracelular monofosfato cíclico de adenosina (AMPc) como um parâmetro essencial para definir a função das células β. 

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Um método para isolamento de ilhotas murino e transplante renal subcapsular

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Medicina

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Análises de célula única Transcriptomic de células endócrinas no pâncreas de rato

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Biologia do Desenvolvimento

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Mouse Modelo de Diabetes Tipo 2: Isolamento de ilhotas pancreáticas e células vivas 2-Photon Imagens de ilhotas intactas

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Um rastreio de baseada em célula de murino ilhotas pancreáticas para produtos químicos ambientais Diabetogenic

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Imunologia e Infecção

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.

Rato Ilhéu de Langerhans isolamento usando uma combinação de colagenase purificada e Protease Neutra

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 ...

Biologia

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Visualização de complexos de Mitophagy endógenos in situ em células beta pancreáticas humanas utilizando o ensaio de ligadura de proximidade

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

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.

Um protocolo de alta eficiência simples para isolamento de Islet pancreático de camundongos

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

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.

Dinâmica de cálcio de imagem em subpopulações de células de ilhotas pancreáticas do rato

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

<meta charset="utf8"></head><body>Simplificando a extração de células endoteliais pancreáticas de camundongo para estudos de doenças

Isolation of Mouse Pancreatic Endothelial Cells

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

<meta charset="utf8"></head><body>Isolamento de células endoteliais pancreáticas de camundongo

This protocol describes the isolation of mouse endothelial cells from whole pancreas.

Isolamento de células endoteliais pancreáticas de camundongo

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and ...

Um Método para Mouse ilhotas pancreáticas Isolamento e intracelular cAMP Determinação

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