Biz bu çalışmada açıklanan protokollere uzman teknik yardım için Renee L. Pasker ve Harpreet K. Brar teşekkür etmek istiyorum. Ayrıca, bize zaman izin laboratuvar üyelerinin desteği ile birlikte, Wisconsin-Madison Üniversitesi Duke Üniversitesi ve Alan D. Attie, Christopher B. Newgard ve danışmanlık kabul ve optimize etmek için gerekli destek istiyoruz açıklanan protokolleri. Özellikle, verimli tartışmalar ve tavsiye için Attie Laboratuarında Hans HOHMEIER, Danhong Lu, ve Helena Winfield Newgard Laboratuvarı ve Mary Rabaglia teşekkür ederim. Bu çalışma NIH hibe DK080845 ve Çocuk Diyabet 594 tarafından desteklenen (MEK) Araştırma Vakfı hibe 17-2011-608

<ol>
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Yazarlar ifşa hiçbir şey yok.

Dünya nüfusunun% 7.7 etkileyebilecek tahmin diyabet yaygınlığı ile, yeni araştırma tekniklerinin gereksinimi hem anlamak ve diyabet 18 tedavisi zorunludur. Mevcut adacık izolasyon in vitro deney için kullanılan bir iyi kurulmuş bir protokoldür ve hafif değişiklikler 11,14,16 ile daha önce sunulmuştur. Insülin salgılanması gibi, cAMP gibi yukarı bileşenleri üzerinde odaklanarak, izole adacık için ortak bir alt bir uygulama olmakla birlikte, insülin üretimini ve sa...

Öglisemi sıkı bakım kontrolsüz tip 1 ve 2 diyabet 1 patoloji tüm özellikleridir nöropati, nefropati ve retinopati, hastalıklarının önlenmesi için zorunludur. Tip 1 ve tip 2 diyabetli hem de azaltılmış β-hücre fonksiyonu ve kütle kan glikozu konsantrasyonlarını 2 de karıştırabilir. Insülin üreten β-hücrelerinin, bozulmuş β-hücresi insülin salgılama ve çevresel insülin türü sinyal 2 diyabet yıkıcı kaybı immün aracılı tip 1 diyabet, birlikte hiperglisemi, dislipidemi ve sonunda ile sonuçlanan, artan hepatik glikoz üretimini teşvik Oysa β-hücrelerinin ayrı ayrı 3 β-hücre kütlesi ve insülin salgılama kapasitesinin kaybı. iki Tip 1 ve tip 2 diyabet ilerlemesinde temel β-hücresi mekanizmaların anlaşılması, bu hastalıkları önlemek ve tedavi etmek için yeni tedavilere yol açacaktır. Vitro ti içindeBöyle INS-1 ve MIN6 gibi ssue kültür modelleri, β-hücre hatları ölümsüzleştirdi spesifik β-hücre fonksiyonlarını anlamak için yararlı bir araç olabilir. Bununla birlikte, adacık içinde farklı hücre tipleri arasında etkileşim kendileri β-hücresi fonksiyonunu regüle olabilir. Örneğin, glukagon parakrin etkisi (α-hücrelerinden salınan) ve insülin salgılanmasını artan ve azalan somatostatin (δ-hücrelerinden salınan), sırasıyla, endokrin tepkisinin 4 hücre, hücre yakınlık önemini ortaya koymaktadır. Ayrıca, β-hücreleri arasındaki ara bağlantılar insülin 5 salınımını mümkün kılmaktadır. Adımlar daha glikoz için izole edilmiş adacık fizyolojik yanıtı çoğaltılmış insülinom çizgileri üreten yapılmıştır, ancak ayrıca, (örneğin, INS-1-türevi 832/13 ve 832/3 hücre çizgileri), glikoz yanıt yine de, normal fare farklıdır 6,7 adacıklarının. Ayrıca, bu klonal insülinoma hücre çizgilerinin tepkiglukagon benzeri peptid-1 (GLP-1), bunların agonistlere karşı birbirinden önemli ölçüde farklı, hem de normal adacık 6 olabilir. Bu nedenle, ölümsüzleştirdi hücre hatları Maddeleri cAMP üretimi üzerinde etkisi olduğu tahlil için en iyi modeli temsil olmayabilir. Insülinom-türevi hücre hatlarının tersine, bütün hayvan modellerinde, sadece β-hücresi fonksiyonu çalışmalarında komplikasyonların kendi kümesi sunar. Endokrin dokusu ile çalışan en büyük zorluklardan biri serbest hormon kesin konsantrasyonu ölçülmesidir. Özel olarak, karaciğer insülin metabolize önemli bir rol oynar ve kan akışı pankreas, karaciğer doğrudan gider. Böylece, plazma insülin ölçümü doğru pankreas kendisi veya insülin salgılanmasının 8&#39;in oranı farklı tedavilerin etkisinden salgılanan insülinin miktarda tasvir olmayabilir. Ayrıca, glukagon renal metabolizması α-adacık hücreleri 9 glukagon çıkış güvenilirliğini sınırlayabilir. Bu nedenle, in vitro deney için primer fare adacıkları izole adacık in vivo koşullarında yapılan ölçümler güzelleştirmek için belli bir uyarana yanıt nasıl daha kesin bir anlaşılmasını sağlar. Fare adacıklar izolasyonu için mevcut protokol (başarısını artırmak için yardımcı olabilir hafif değişiklikler) gruplarının bir dizi 10,11 tarafından kullanılan köklü bir protokoldür. Buna ek olarak, cAMP üretiminin belirlenmesi β-hücrelerinin iç salgı bezi yanıt doğrudan bir-okuma sağlar. CAMP ölçümü, protein içeriği ve insülin salgılanması ile bağlantılı olarak da β-hücre fonksiyonu bir kusur cAMP 10 proksimal veya distal yatıyor belirlemek için yardım, aynı cAMP numune hazırlık dan ölçülebilir. Bu protokol son cAMP içeriği ve insülin salgılanması uygulama aralarında, ilaç ve diyet bileşenlerinin etkisini anlamak için çok güçlü bir araç olabilirs, cAMP ve insülin salgılanması üzerinde. Tek başına glikozdan uyarılmaya ek olarak, diğer bileşikler, cAMP ve insülin salgılanması 10,11 değişiklikleri ölçmek için kullanılabilir. İzole adacık insülin, glukagon ve bu tür somatostatin gibi diğer hormonlar,, hem de sitokinler, eikosanoidler ve siklik adenozin monofosfat ikinci tahlil birincil hormon olmasına rağmen, son olarak, aynı zamanda geçici bir uyarma deneyi ile ya da miktarının ölçülmesi ile, ya ölçülebilir kültür ortamında 12 kendi seviyeleri. Son olarak, diğer birçok uygulamalar, alt doku adacığı transplantasyonu, PCR ya da mikro-dizi analizi, nicel gerçek zaman RNA izolasyonu, bir protein olarak, takip edilebilir, böylece dış Bu yazının kapsamı, tarif edilen kolajenaz izolasyon yöntemi ile doku adacığı izolasyonu adacık koruma sağlar, ancak Western lekeleme, adacık yerleştirmesi ve immünofloresan görüntüleme ve adacık hücresi Repli bir ölçüsü olarak, [3H]-timidin dahil edilmesi için izolasyonÖnceki JoVE makalelerinde 13-16 &#39;de tarif edilmiştir bazıları katyondur. Genel olarak, protokolde tarif adacık izolasyon prosedürü takip tedavilerin geliştirilmesi ve β-hücre fonksiyonu artırmaya yönelik ilaç keşfi teşvik için önemli ve yararlı bilgiler içeren bir araştırmacı sağlayabilir.

<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

Kontrolsüz glisemi diabetes mellitus bir özelliğidir ve nöropati, nefropati ve retinopati gibi hastalıklarının teşvik etmektedir. Diyabet, immün-aracılı tip 1 ve obezite bağlantılı tip 2 hem de diyabet patofizyolojisi ve tedavi mekanizmalarını ortaya koymaya yönelik çalışmaların artan yaygınlığı ile kritik bir önem taşımaktadır. Langerhans pankreatik adacıkların β-hücrelerinin uygun bir şekilde, yüksek kan şekeri konsantrasyonlarına yanıt olarak insülin salgılayan sorumludur. Glikoz ve diğer besinler, β-hücrelerinin aynı zamanda belirli hormonlar tarafından uyarılır Buna ek olarak, hücre içi siklik adenozin monofosfat üretimini artırmak β-hücresi reseptörleri üzerinde bir yemek ve hareket yanıt olarak bağırsak salgılanan inkretinler, (adlandırılır cAMP). Azalmış β-hücre fonksiyonu, kütle ve iç salgı bezi yanıt tip 2 diyabet patofizyolojisi katkıda iyi anlaşılmış olup, aynı zamanda giderek wi bağlantılı ediliyorinci tip 1 diyabet. Mevcut fare adacık izolasyon ve cAMP tespit protokolü hastalığın ilerlemesini ve terapötik müdahaleler, hücre içi cAMP üretiminin modülasyonu yoluyla hareket reseptörlerini inkretindir reseptörlerinin aracılık ettiği veya ilgili olduğu özellikle teşvik mekanizmaları tanımlamak yardımcı bir araç olabilir. Sadece cAMP ölçümleri tarif edilecek olmasına rağmen, tarif edilen doku adacığı izolasyonu protokolü de glikoz insülin salgılanmasını, [3H]-timidin katılması, protein bolluğu ve mRNA ifadesi uyarılmış de dahil olmak üzere diğer pek çok alt uygulamalar için izin temiz bir hazırlık oluşturur.

Izolasyon sırasında yüksek adacık verim sağlamak için, protokolde belirtilen cerrahi teknikler yakından takip edilmelidir. Burada sunulan teknikleri her laboratuvara uygun olacak olsa da, başarılı bir izolasyona yol açacak bir kaç kritik adımlar vardır. Safra kanalı kolayca erişilebilir hale getirmek için, bu organlar farenin sağ tarafına (Şekil 1) yer değiştirebilir önerilir. Ayrıca, bu daha az ağırlık kısıtlayan genişleme olacağından pankreas direnci küçük bir miktar...

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

Langerhans adacıkları izole edilmiş fare kullanılarak in vitro β-hücre fonksiyonu denenmesi diyabet patofizyolojisi ve terapötik çalışmada önemli bir bileşenidir. Çok sayıda alt-uygulama mevcut olmakla birlikte, bu protokol, spesifik olarak β-hücresi fonksiyonunu belirleyen önemli bir parametre olarak hücre içi siklik adenozin monofosfat (cAMP) ölçümünü açıklamaktadır.

Fare Pankreas Adacık İzolasyonu ve hücre içi cAMP Belirlenmesi için Bir Yöntem

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

65.1K Görüntüleme.  University of Wisconsin-Madison.  Langerhans adacıkları izole edilmiş fare kullanılarak in vitro β-hücre fonksiyonu denenmesi diyabet patofizyolojisi ve terapötik çalışmada önemli bir bileşenidir. Çok sayıda alt-uygulama mevcut olmakla birlikte, bu protokol, spesifik olarak β-hücresi fonksiyonunu belirleyen önemli bir parametre olarak hücre içi siklik adenozin monofosfat (cAMP) ölçümünü açıklamaktadır. 

Video: 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.

Murin Islet İzolasyon ve subkapsüler Böbrek Nakli Bir Yöntem

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

Tıp

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.

Fare pankreatik endokrin hücreleri tek hücre Transcriptomic analizleri

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

Gelişim Biyolojisi

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; Tip 2 Diyabet Fare Modeli: Bozulmamış Islets Of Pankreas Adacık İzolasyonu ve Canlı hücre 2-Foton Görüntüleme

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.

Bir fare pankreas adacık hücre tabanlı tarama diyabetojenik çevresel kimyasallar için

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 ...

İmmünoloji ve Enfeksiyon

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.

Langerhans İzolasyonu Fare Islet Saflaştırılmış Collagenase ve Nötr Proteaz bir kombinasyonu kullanılarak

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 ...

Biyoloji

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.

Yakınlık ligasyon assay kullanarak ınsan pankreatik Beta hücrelerinde situ Içinde endojen Mitophagy kompleksleri görselleştirme

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.

Farelerden Pankreas Adacık İzolasyon için Basit Bir Yüksek Verimlilik Protokolü

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.

Fare Pankreas Adacık Hücrelerinin Alt Popülasyonlarında Görüntüleme Kalsiyum Dinamiği

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>Hastalık Çalışmaları için Fare Pankreas Endotel Hücre Ekstraksiyonunun Kolaylaştırılması

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>Fare Pankreas Endotel Hücrelerinin İzolasyonu

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

Fare Pankreas Endotel Hücrelerinin İzolasyonu

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 ...

Fare Pankreas Adacık İzolasyonu ve hücre içi cAMP Belirlenmesi için Bir Yöntem

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Murin Islet İzolasyon ve subkapsüler Böbrek Nakli Bir Yöntem

Fare pankreatik endokrin hücreleri tek hücre Transcriptomic analizleri

Lepr

Bir fare pankreas adacık hücre tabanlı tarama diyabetojenik çevresel kimyasallar için

Langerhans İzolasyonu Fare Islet Saflaştırılmış Collagenase ve Nötr Proteaz bir kombinasyonu kullanılarak

Yakınlık ligasyon assay kullanarak ınsan pankreatik Beta hücrelerinde situ Içinde endojen Mitophagy kompleksleri görselleştirme

Farelerden Pankreas Adacık İzolasyon için Basit Bir Yüksek Verimlilik Protokolü

Fare Pankreas Adacık Hücrelerinin Alt Popülasyonlarında Görüntüleme Kalsiyum Dinamiği

Fare Pankreas Endotel Hücrelerinin İzolasyonu

Fare Pankreas Endotel Hücrelerinin İzolasyonu