This work was funded by NIH grants R01 DK123292, T32 DK108736, UC4 DK104194, UG3 DK122638, and P01 AI042288. This research was performed with the support of the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project sponsored by JDRF (nPOD: 5-SRA-2018-557-Q-R), and The Leona M. &#38; Harry B. Helmsley Charitable Trust (Grant #2018PG-T1D053). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org/for-partners/npod-partners/. Thank you to Dr. Kevin Otto, University of Florida, for providing the vibratome used to generate mouse slices.&#160;

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The authors declare no competing interests.

The objective of this protocol is to explicate the generation of pancreas slices and the procedures needed to employ the slices in functional and immunological studies. There are many benefits to using live pancreatic slices. However, there are several critical steps that are essential for the tissue to remain viable and useful during the described experiment protocols. It is imperative to work quickly. The length of time between injecting the pancreas and generating the slices on the vibratome should be minimized to mai...

Involvement of the pancreas is pathognomonic to diseases such as pancreatitis, T1D, and T2D1,2,3. The study of function in isolated islets usually involves removal of the islets from their surrounding environment4. The live pancreatic tissue slice method was developed to allow for the study of pancreatic tissue while maintaining intact islet microenvironments and avoiding the use of stressful islet isolation procedures5,6,7. Pancreatic tissue slices from human donor tissue have been successfully used to study T1D and have demonstrated processes of beta cell loss and dysfunction in addition to immune cell infiltration8,9,10,11,12,13. The live pancreatic tissue slice method can be applied to both mouse and human pancreatic tissue5,6,8. Human pancreatic tissue slices from organ donor tissues are obtained through a collaboration with the Network for Pancreatic Organ Donors with Diabetes (nPOD). Mouse slices can be generated from a variety of different mouse strains.
This protocol will focus on non-obese diabetic-recombination activating gene-1-null (NOD.Rag1-/-) and T cell receptor transgenic (AI4) (NOD.Rag1-/-.AI4&#945;/&#946;) mouse strains. NOD.Rag1-/- mice are unable to develop T and B cells due to a disruption in the recombination-activating gene 1 (Rag1)14. NOD.Rag1-/-.AI4&#945;/&#946; mice are used as a model for accelerated type 1 diabetes because they produce a single T cell clone that targets an epitope of insulin, resulting in consistent islet infiltration and rapid disease development15. The protocol featured here describes procedures for functional and immunological studies using live human and mouse pancreatic slices through the application of confocal microscopy approaches. The techniques described herein include viability assessments, islet identification and location, cytosolic Ca2+ recordings, as well as staining and identification of immune cell populations.

<table><tbody><tr><td>#3 Style Scalpel Handle</td><td>Fisherbrand</td><td>12-000-163</td><td></td></tr><tr><td>1 M HEPES</td><td>Fisher Scientific</td><td>BP299-100</td><td>HEPES Buffer, 1M Solution</td></tr><tr><td>10 cm Untreated Culture Dish</td><td>Corning</td><td>430591</td><td></td></tr><tr><td>10 mL Luer-Lok Syringe</td><td>BD</td><td>301029</td><td>BD Syringe with Luer-Lok Tips</td></tr><tr><td>27 G Needle</td><td>BD</td><td>BD 305109</td><td>BD General Use and PrecisionGlide Hypodermic Needles</td></tr><tr><td>35 mm coverglass-bottom Petri dish</td><td>Ibidi</td><td>81156</td><td>&amp;micro;-Dish 35 mm, high</td></tr><tr><td>50 mL syringe</td><td>BD</td><td>309653</td><td></td></tr><tr><td>8-well chambered coverglass</td><td>Ibidi</td><td>80826</td><td>&amp;micro;-Slide 8 Well</td></tr><tr><td>APC anti-mouse CD8a antibody</td><td>Biolegend</td><td>100712</td><td></td></tr><tr><td>BSA</td><td>Fisher Scientific</td><td>199898</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5670</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>C7902</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (dihydrate)</td></tr><tr><td>Compact Digital Rocker</td><td>Thermo Fisher Scientific</td><td>88880020</td><td></td></tr><tr><td>Confocal laser-scanning microscope</td><td>Leica</td><td>SP8</td><td>Pinhole&amp;thinsp;= 1.5-2 airy units; acquired with 10x/0.40 numerical aperture HC PL APO CS2 dry and 20x/0.75 numerical aperture HC PL APO CS2 dry objectives at 512&amp;thinsp;&amp;times;&amp;thinsp;512 pixel resolution</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;</td></tr><tr><td>ddiH2O</td><td></td><td></td><td></td></tr><tr><td>Dithizone</td><td>Sigma-Aldrich</td><td>D5130-10G</td><td></td></tr><tr><td>DMSO</td><td>Invitrogen</td><td>D12345</td><td>Dimethyl sulfoxide</td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>2805</td><td></td></tr><tr><td>Falcon 35 mm tissue culture dish</td><td>Corning</td><td>353001</td><td>Falcon Easy-Grip Tissue Culture Dishes</td></tr><tr><td>FBS</td><td>Gibco</td><td>10082147</td><td></td></tr><tr><td>Feather No. 10 Surgical Blade</td><td>Electron Microscopy Sciences</td><td>7204410</td><td></td></tr><tr><td>fluo-4-AM</td><td>Invitrogen</td><td>F14201</td><td>cell-permeable Ca&lt;sup&gt;2+&lt;/sup&gt; indicator</td></tr><tr><td>Gel Control Super Glue</td><td>Loctite</td><td>45198</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11049-10</td><td></td></tr><tr><td>Hardened Fine Scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025092</td><td>Hanks Balanced Salt Solution</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td>C&lt;sub&gt;8&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;S</td></tr><tr><td>Ice bucket</td><td>Fisherbrand</td><td>03-395-150</td><td></td></tr><tr><td>Isoflurane</td><td>Patterson Veterinary</td><td>NDC 14043-704-05</td><td></td></tr><tr><td>Johns Hopkins Bulldog Clamp</td><td>Roboz Surgical Store</td><td>RS-7440</td><td>&amp;nbsp;Straight; 500-900 Grams Pressure; 1.5&quot; Length</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>34705</td><td>Kimtech Science&amp;trade; Kimwipes&amp;trade; Delicate Task Wipers, 2-Ply</td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td>This kit contains the calcein-AM live cell dye.</td></tr><tr><td>Low glucose DMEM</td><td>Corning</td><td>10-014-CV</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma</td><td>M9272</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (hexahydrate)</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>M2773</td><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (heptahydrate)</td></tr><tr><td>Magnetic Heated Platform</td><td>Warner Instruments</td><td>PM-1</td><td>Platform for imaging chamber for dynamic stimulation recordings</td></tr><tr><td>Microwave</td><td>GE</td><td>JES1460DSWW</td><td></td></tr><tr><td>Nalgene Syringe Filter</td><td>Thermo Fisher Scientific</td><td>726-2520</td><td></td></tr><tr><td>No.4 Paintbrush</td><td>Michaels</td><td>10269140</td><td></td></tr><tr><td>Open Diamond Bath Imaging Chamber</td><td>Warner Instruments</td><td>RC-26</td><td>Imaging chamber for dynamic stimulation recordings</td></tr><tr><td>Oregon Green 488 BAPTA-1-AM</td><td>Invitrogen</td><td>O6807</td><td>cell-permeable Ca&lt;sup&gt;2+&lt;/sup&gt; indicator</td></tr><tr><td>Overnight imaging chamber</td><td>Okolab</td><td>H201-LG</td><td></td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>20012050</td><td>To make agarose for slice generation</td></tr><tr><td>PE-labeled insulin tetramer</td><td>Emory Tetramer Research Core</td><td>sequence YAIENYLEL</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P5405</td><td>KCl</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P5655</td><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Razor Blades</td><td>Electron Microscopy Sciences</td><td>71998</td><td>For Vibratome; Double Edge Stainless Steel, uncoated</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>SeaPlaque low melting-point agarose</td><td>Lonza</td><td>50101</td><td>To make agarose for slice generation</td></tr><tr><td>Slice anchor</td><td>Warner Instruments</td><td>64-1421</td><td></td></tr><tr><td>Slice anchor (dynamic imaging)</td><td>Warner Instruments</td><td>640253</td><td>Slice anchor for dynamic imaging chamber</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S5886</td><td>NaCl</td></tr><tr><td>Sodium phosphate monohydrate</td><td>Sigma</td><td>S9638</td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;(monohydrate)</td></tr><tr><td>Soybean Trypsin Inhibitor</td><td>Sigma</td><td>T6522-1G</td><td>Trypsin inhibitor from Glycine max (soybean)</td></tr><tr><td>Stage Adapter</td><td>Warner Instruments</td><td>SA-20MW-AL</td><td>To fit imaging chamber for dynamic stimulation recordings on the microscope stage</td></tr><tr><td>Stage-top incubator</td><td>Okolab</td><td>H201</td><td></td></tr><tr><td>Stereoscope</td><td>Leica</td><td>IC90 E MSV266</td><td></td></tr><tr><td>SYTOX Blue Dead Cell Stain</td><td>Invitrogen</td><td>S34857</td><td>blue-fluorescent nucleic acid stain</td></tr><tr><td>Transfer Pipet</td><td>Falcon</td><td>357575</td><td>Falcon&amp;trade; Plastic Disposable Transfer Pipets</td></tr><tr><td>Valve Control System</td><td>Warner Instruments</td><td>VCS-8</td><td>System for dynamic stimulation recordings</td></tr><tr><td>Vibratome VT1000 S</td><td>Leica</td><td>VT1000 S</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>FSGPD02</td><td>Fisherbrand Isotemp General Purpose Deluxe Water Bath GPD 02</td></tr></tbody></table>

observing islet function and islet-immune cell interactions in live pancreatic tissue slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This protocol will yield live pancreatic tissue slices suitable for both functionality studies and immune cell recordings. Slice appearance in both brightfield and under reflected light are shown in Figure 1A,B. As discussed, islets can be found in slices using reflected light due to their increased granularity that occurs because of their insulin content (Figure 1C) and are clearly observed compared to the background tissue when reflected light...

Watch this Scientific Journal Video about Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices at JoVE.com

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

observing-islet-function-islet-immune-cell-interactions-live

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JoVE Journal

Immunology and Infection

4.0K Views.  University of Florida. This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions. 

Video: Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

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.

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

Medicine

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

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.

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, ...

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.

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

Biology

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

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.

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.

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

Pancreatic Islet Embedding for Paraffin Sections

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

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

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

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Slicing Human Pancreas for Islet Function Research

Generating Human Pancreatic Tissue Slices to Study Endocrine and Exocrine Pancreas Physiology

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the pancreas endocrine and exocrine physiology and interplay. Much has been learned from the study of isolated pancreatic islets, but this prevents examining their function and interactions in the context of the whole tissue. Pancreas slices provide a unique opportunity to explore the physiology of normal, inflamed, and structurally damaged islets within their native environment, in turn allowing the study of interactions between endocrine and exocrine compartments to better investigate the complex dynamics of pancreatic tissue. Thus, the adoption of the living pancreas slice platform represents a significant advancement in the field. This protocol describes how to generate living tissue slices from deceased organ donors by tissue embedding in agarose and vibratome slicing as well as their utilization to assess functional readouts such as dynamic secretion and live cell imaging.

Author Spotlight: Advancing Type 1 Diabetes Research Using Innovative Pancreatic Slice Platforms

This protocol describes how to generate human pancreas slices from deceased organ donors to study cell function under near-physiological conditions. This innovative approach enables the investigation of normal and structurally damaged islets and the intricate interplay between endocrine and exocrine compartments.

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the ...