Name: imaging calcium dynamics in subpopulations of mouse pancreatic islet cells
Uploaded: 2019-11-26T12:00:00
Description: Watch this Scientific Journal Video about Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells at JoVE.com

AH was a recipient of a Diabetes UK PhD Studentship, EV was supported by the OXION-Wellcome Trust Training Programme, AIT held an Oxford Biomedical Research Council postdoctoral fellowship.

All methods described here were developed in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986) and the University of Oxford ethical guidelines.
1. Isolate mouse pancreatic islets
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	<li>Prepare the culture medium and the isolation solution.
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		<li>Make up the culture medium: RMPI1640 (see Table of Materials), supplemented with 10% of fetal calf serum, 100 units/mL penicillin, 100 &#956;g/mL streptomycin. Dispense the medium into two 60 mm ...

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There are three stages in the protocol that are critical for overall success. Successful injection of the Liberase enzyme into the bile duct determines not just the quantitative success of the isolation procedure but also impacts the quality of the isolated islets. Uninflated pancreata may result in lack of some important metabolic responses in the isolated islets. Secondly, the loading of the dye/the expression of the sensor defines the signal-to-noise ratio of the time-lapse recording. Signals are absent or attenuated ...

The purpose of the method is to image real-time changes in cytosolic calcium concentration ([Ca2+]cyt) in minor subpopulations of pancreatic islet cells. This allows uncovering the mechanisms governing hormone secretion in these cells, revealing details about the cross-talk between different cell types and, potentially, introducing a populational dimension into the larger picture of islet signaling.
Islets consist of several cell types. Besides the more well-known insulin-secreting &#946;-cells, there are at least two subpopulations that are also critical in regulating blood glucose3. &#945;-Cells (that make up around 17% of islet cells) secrete glucagon when blood glucose gets too low, which signals for release of glucose into the bloodstream from depots in the liver. Excessive glucagon levels (hyperglucagonemia) and impaired control of glucagon-release accompany (and, technically, can contribute to) the prediabetic condition of impaired insulin sensitivity4. &#948;-Cells (around 2%) secrete somatostatin in response to glucose elevation. This ubiquitous peptide hormone is likely to be present at high concentrations in the vicinity of &#945;- and &#946;-cells within islets, which has a strong Gi receptor-mediated attenuating effect on both glucagon and insulin secretion.
&#945;-Cells and &#948;-cells share a large part of the glucose-sensing machinery with their close lineage relatives, &#946;-cells. All three cells types are equipped with ATP-sensitive K+ channels, elaborate metabolic sensors5 that control the plasma membrane potential of these excitable cells. At the same time, secretion of insulin, somatostatin and glucagon is regulated differently by glucose. Imaging of Ca2+ dynamics in the two minor subpopulations of islet cells can therefore provide an insight into the cross-talk between blood glucose and islet secretory output.
Early attempts of monitoring the excitability of &#945;- and &#948;-cells using patch-clamp electrophysiology were soon followed by imaging of Ca2+ in single &#945;- and &#948;-cells. The identity of cells in these experiments was verified via a posteriori staining with anti-glucagon or anti-somatostatin antibodies. These efforts were frequently hampered by the finding that islet cells behave very differently within the islet and as single cells. Although &#946;-cells may appear to be the main benefactors of the islet arrangement (due to their overwhelming majority that underlies their strong electrical coupling), the main discrepancy was, surprisingly, found in &#945;-cells. Within the intact islet, these cells are constantly and persistently activated at low glucose, which is only true for around 7% of single dispersed &#945;-cells6. Reporting the activity of &#945;- and &#948;-cells within intact islets is therefore believed to represent a closer approximation of in vivo conditions.
In general, there are two ways of reporting Ca2+ dynamics specifically from the &#945;-cell or &#948;-cell subpopulations: (i) expressing a genetically encoded Ca2+ sensor via a tissue-specific promoter or (ii) using marker compounds. The more elegant former approach adds the substantial advantage of true 3D imaging and hence studying of cell distribution within the islet. It cannot however be applied for intact human islet material. Another potential concern is the &#39;leakiness&#39; of the promoter, particularly when the &#946;-/&#945;-cell transdifferentiation or &#945;-cell response to high glucose is in place. The latter approach can be used with freshly isolated tissue including human samples or cultured islets. The data, however, is collected solely from the peripheral layer of islet cells, as delivering the dye/marker molecule in deeper layers without altering the islet architecture is challenging. An unexpected advantage of the latter approach is the compatibility with wide-field imaging mode, which allows scaling up the experiments to simultaneous imaging of tens or hundreds of islets (i.e., thousands to tens of thousands of cells).
Calcium is imaged in vivo using genetically encoded GCaMP7 (or pericam8) family sensors, which are variants of circularly permutated green fluorescent protein (GFP) fused to the calcium-binding protein calmodulin and its target sequence, M13 fragment of myosin light chain kinase7,9. GCaMPs have superb signal-to-noise ratios in the range of nanomolar Ca2+ concentrations and a high 2-photon cross-section, which makes them an ideal choice for in vivo work10,11. The challenging aspect of using recombinant sensors is their delivery into the cells. Heterologous expression requires using a viral vector and multi-hour ex vivo culturing, which frequently raises concerns regarding potential de-differentiation or deterioration of cell functions. Although mouse models pre-engineered to express GCaMP address this problem, they add new challenges by increasing the lead time substantially and limiting the work to a non-human model. Very high sensitivity to changes of intracellular pH is another adverse side of protein-based sensors12, which is, however, less of a problem for sensing oscillatory signals, such as Ca2+.
The advantage of trappable dyes (such as green fluorescent Fluo4) is that they can be loaded into freshly isolated tissue within around an hour. Predictably, trappable dyes have lower signal-to-noise ratios and (much) lower photostability than their recombinant counterparts. We cannot confirm13 the reports of toxicity of the trappable dyes14, however, dye overloading is a frequent problem.
Red recombinant Ca2+ sensors based on circular permutation have been evolving rapidly since 201115, and most recent developments present a strong competition to GCaMPs16 for tissue imaging, given higher depth of penetration of red light. Commercially available red trappable dyes can be used reliably for single-cell imaging but, on the tissue level, cannot compete well with the green analogs.
There is seemingly very little choice of imaging technology for experiments in tissue where out-of-focus light becomes a critical problem. The confocal system provides acceptable single-cell resolution by cancellation of the out-of-focus light with any objective on the NA above 0.3 (for the case of GCaMP6) or 0.8 (trappable dye). In a technical sense, a conventional confocal microscope can be used for simultaneous imaging of [Ca2+]cyt from hundreds (GCaMP) or tens of islets (trappable dye). The only realistic alternative to confocal mode in case of 3D expression of the sensor in tissue is perhaps light-sheet microscopy.
Things are slightly different for the case when the sensor is expressed in the peripheral layer of cells within the islet tissue. For bright recombinant sensors that have a vivid intracellular expression pattern, using a wide-field imaging mode with a low-NA objective may provide sufficient quality and reward the researcher with a substantial increase in the field of view area and hence the throughput. A wide-field system provides poorer spatial resolution, as the out-of-focus light is not cancelled; therefore, imaging tissue with high-NA (low depth of field) objectives is less informative, as the single-cell signal is vastly contaminated by neighboring cells. The contamination is much smaller for low-NA (high depth of field) objectives.
There are tasks, however, for which high throughput and/or sampling rate become a critical advantage. &#945;- and &#948;-cells exhibit substantial heterogeneity, which creates a demand for high sample sizes to reveal the contribution of the subpopulations. Wide-field imaging is fast and more sensitive, with an industrial-scale large field-of-view system imaging hundreds (GCaMP) or tens (Fluo4) of islets at the same signal-to-noise ratio as the confocal experiments on ten or a single islet, respectively. This difference in throughput makes the wide-field system advantageous for populational imaging with a single-cell resolution, which can be especially critical for small subpopulations such as the &#948;-cell one. Likewise, attempts to reconstruct electrical activity from Ca2+ spiking17 would benefit from the higher sampling rate provided by a wide-field imaging mode. At the same time, several &#34;niche&#34; problems like the activity of pancreatic &#945;-cells upon stimulation of the dominating &#946;-cell subpopulation, require the use of a confocal system. A factor that influences the decision towards confocal mode is the presence of substantial contaminating signal from the &#946;-cell subpopulation.
Although using hormone-specific antibody staining to verify the identity of the cells after the imaging experiments is still an option, minor cell subpopulations can be identified using functional marker compounds, such as adrenaline and ghrelin that were shown to selectively stimulate Ca2+ dynamics in &#945;-18 and &#948;-cells19,20, respectively.
The analysis of time-lapse imaging data aims to provide information beyond trivial pharmacology, such as populational heterogeneity, correlation and interaction of different signals. Conventionally, imaging data is analyzed as intensity vs. time and normalized to the initial fluorescence (F/F0). Baseline correction is frequently needed, due to the bleaching of the fluorophore signal or contamination by changes in autofluorescence or pH (typically induced by millimolar levels of glucose12). Ca2+ data can be analyzed in many different ways, but three main trends are to measure changes in the spike frequency, the plateau fraction, or area under the curve, computed vs. time. We found the latter approach advantageous, especially in application to heavily undersampled confocal data. The advantage of the pAUC metric is its sensitivity to both changes in signal frequency and amplitude, whereas computing the frequency requires a substantial number of oscillations21, which is hard to attain using conventional imaging. The limiting factor of pAUC analysis is its high sensitivity to baseline changes.

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			<td height="21" style="height:21px;width:216px;">Fetal bovine serum</td>
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			<td height="21" style="height:21px;width:216px;">Fluo4</td>
			<td style="width:277px;">ThermoFisher (Life Technologies)</td>
			<td style="width:119px;">&#160; F14201</td>
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			<td height="42" style="height:42px;width:216px;">GCaMP6f, in (human type 5) adenoviral vector</td>
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			<td height="21" style="height:21px;width:216px;">Hanks&#39; solution&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Liberase</td>
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			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
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			<td style="width:119px;">15140122</td>
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			<td style="width:277px;">ThermoFisher (GibCo, Life Technologies)</td>
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			<td height="42" style="height:42px;width:216px;">Zeiss LSM510-META confocal system</td>
			<td style="width:277px;">Carl Zeiss</td>
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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.

Islets load fairly well with the trappable dyes (Figure 1A), unless the lipid composition of the membrane has been affected (e.g., by chronic exposure to fatty acids). The human adenovirus type 5 (Ad5) vector also targets all islet cells (Figure 1B). Problems may arise when more than one recombinant sensor is being expressed in the same cell. Furthermore, islets are typically very well immobilized using the technology described ...

Watch this Scientific Journal Video about Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells at JoVE.com

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic 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.

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

The protocol allows rapid isolated function of small subpopulations of cells within pancreatic islets of Langerhans. The advantages of this technique include the real-time nature and high statistical power, and hence high reproducibility, of the acquired data. To immobilize the islets for imaging, assemble an imaging chamber for an inverted microscope and place a glass cover slip inside the chamber.Make sure that the glass chamber interface is watertight and confirm that the cover slip is within the reach of the microscope objective. Next, cut 20 by 20 millimeter squares of fine and coarse mesh and use 45 to 50 micrometer pieces of thick sticky tape to create two spacer walls on a piece of fine mesh. Immerse the coarse mesh and a weight in a 35 millimeter Petri dish of imaging solution, and place the fine mesh under a dissecting microscope.Turn the fine mesh with the spacer walls upside down and the spacers facing upwards. And use a p20 pipette to transfer several islets between the two spacers. Using watchmakers'forceps, place the mesh upside down inside the imaging chamber of the inverted microscope so that the spacers face downward to sit directly on the chamber cover slip, trapping the islets between the spacers and the mesh in the middle of the cover slip.Take care that the mesh is well hydrated without containing excessive volumes of solution which would provoke the lateral motion of the sample. Then place the coarse mesh and the weight on top of the fine mesh in the chamber, and add imaging solution to the chamber. Once the islets have been immobilized, select the imaging mode and objective on the inverted microscope and place the chamber with the islets on the temperature-controlled stage of the microscope.After setting the perfusion, position the inflow lower than the outflow within the chamber and set the outflow flux to be greater than the inflow flux. Ensure that the outflow has minimal contact with the solution so that it removes the solution in multiple sequential small droplets, avoiding long intervals of continuous solution removal. Next, initiate the perfusion with imaging solution containing three millimolar glucose, and select the light path and filters for imaging green fluorophores.Then run live imaging to set up the imaging parameters and adjust the view to capture the islets of interest. To optimize the signal-to-noise ratio of the image, adjust the excitation light intensity, the exposure time, and the binning, ensuring that the settings allow a distinct visualization of each cell within the islets at the minimal possible light intensity and exposure. Then image the islets at 0.1 to five hertz, checking the quality of the acquired data as it is captured.When the alpha cell activity is detectable, use an online chart of the signal dynamics within the acquisition software as possible and add adrenaline or glutamate into the bath solution for two to five minutes. A rapid jump in calcium followed by a slowdown or cancellation of the alpha cell oscillations will be observed. Next, add ghrelin, which has been recently reported to activate delta cells selectively.A rapid reversible increase in calcium will be observed in a small subpopulation of islet cells. Then add 10 millimolar glucose. A coordinated oscillatory response in the beta cell subpopulation will be observed.Note also the responses of cells that were previously activated by adrenaline or glutamate and ghrelin. After saving the images, import the data into an appropriate data analysis software program and normalize the raw fluorescence intensity data to the initial value of fluorescence. If the cell to cell variability fluorescence intensity data set is still substantial, define a control region for the range of time during which the control solution was applied.If the control region has a clear non-oscillatory signal, assume that the fluorescence intensity returned to baseline after each application of the control solution. To correct the time lapse data for each cell, split the data into segments separated by the points at which the control solution was added and apply linear correction to each segment. If the control range has clear oscillations or additional factors are present, use a spike detection algorithm.To measure the frequency of calcium spikes and the response to the addition of agonists and antagonists, split the recording into equal time intervals, count the spikes within the interval, and normalize to the interval duration to compute the time course of the partial frequency. Alternatively, set the threshold and compute the plateau fraction for each of the intervals. The fraction indicates the percentage of time within the interval that the cell spent in the excited state.Or compute the partial area under the curve for each of the intervals. Unless the lipid composition of the membrane has been affected, islets load fairly well with tropical dyes. The human adenovirus type 5 vector also successfully targets islet cells.Calcium spiking in alpha cells can be readily detected at low glucose levels, and there is a high cell-by-cell correlation between the activity at low glucose and the response to adrenaline and glutamate. Ghrelin activates some adrenaline responsive cells at low glucose but has no effect on calcium dynamics in most of the cells that are activated by low glucose. When analyzed in terms of partial frequency, adrenaline or ghrelin-stimulated cells display a substantial increase under the all-or-nothing conditions, although the overall changes between basal spiking and the adrenaline effect are subtle.In contrast, the partial area under the curve is sensitive to the changes introduced by adrenaline in all of the cells even when the basal activity is high. Make sure all the meshes are in contact with imaging solution and take care to position the tissue reasonably densely to avoid air bubbles. The method can be expanded to include the artificial intelligence for differentiating between the cell types.The pharmacological markers can be replaced by the pattern recognition technology, thereby reducing the recording time.

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Research

JoVE Journal

Biology

Murine Pancreatic Islet Isolation

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

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

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

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

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

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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 ...