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.

<ol>
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The Authors declare no conflicts of interest.

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 in overloaded islets. Lastly, successful and dense positioning of the tissue inside the imaging chamber is a defining moment for meaningful and analyzable experiments. Poorly positioned or moving tissue results in wasted experimental time and/or unclear data.
The method can be modified to account for multiple signals (using the confocal system) and multiple groups of islets (e.g., of different genotype). Imaging of multiple signals assumes delivery of a second sensor into each cell of the islet, spectrally compatible with the Ca2+ reporter (such as a pH sensor SNARF5f26,27). To that end, islets can be co-loaded/co-infected with Ca2+ and pH sensors, which are then sequentially imaged within each time-frame.
Imaging the signal in groups of islets with single-cell resolution requires using a wide field-of-view objective. The objective is likely to have lower magnification and numerical aperture (NA), thereby reducing the spatial resolution. Due to the increased depth-of-focus of the low-NA objective, imaging can be performed on a wide-field system. The disadvantages of this arrangement are cell-cell cross-contamination of the light signal and reduced ability to image the 3D signal (e.g., mice expressing the Ca2+ sensor under the insulin promoters). At the same time, signal expressed from the surface islet cells can be perfectly resolved with high temporal resolution from groups including tens to hundreds of islets18.
Although it may sound unpleasant but performing image analysis and numerical data analysis in separate software packages is a good idea. At the current time, ImageJ/FIJI dominates scientific image analysis. The most popular environments for scientific coding are Python and Matlab, yet there are also known efforts to analyze the Ca2+ data in R28. Best usability is provided by more niche packages like IgorPro. Our choice is to prototype in Matlab/Python and then implement the code in IgorPro for &#39;pipeline&#39; use. Adapting signal analysis packages for electrophysiology (e.g., Clampfit, Neuroexplorer) for analytical needs can be useful for single-cell imaging but is difficult to scale up. Many options provided by such packages are not applicable for islet imaging because of low sampling rate.
It is important to remember that this methodology is limited by a number of factors. Firstly, as mentioned above, imaging is largely based on undersampling the data, meaning it does not indicate and therefore cannot be directly compared to electrical activity of the cell. Secondly, the data comes from the islet periphery and does not reflect important coupling processes that are, generally speaking, three-dimensional. Thirdly, the level of loading/expression affects the perception of the sensor intensity. Lastly, activation of less well-researched islet cell subpopulations (e.g., PP cells &#38; &#1013;-cells) by the marker compounds cannot be ruled out, although due to the low numbers of these cells within the islet any potential contamination will be minimal.
The method is a true &#39;champion&#39; in terms of visual effect, as the oscillatory processes deliver a strong impression of a genuinely living tissue. Applied to minor cell subpopulations, the method probes the function of each one reliably, allowing identification of subgroups and reflecting the heterogeneity.
Calcium dynamics has been studied in pancreatic islet &#946;-cells for over 40 years, mostly driven by the progress in acquisition/detection technology. Early studies used atomic absorption spectroscopy29, but it was not until the arrival of fluorescent Ca2+ sensors30 that detailed kinetics could be resolved in individual islet cells, using photometry31,32,33. Soon after that, the spatial component of Ca2+ kinetics was improved as Ca2+ imaging34,35,36 became a routine technology, thanks to the then newly available charge-coupled device (CCD) detectors. The problem of out-of-focus light, that hampered imaging the signal from individual cells within the tissue, was then resolved in the mid-1990s via laser scanning confocal microscopy (LSCM)37 and total internal reflection fluorescence microscopy (TIRFM)38. Both methods, complemented by the arrival of a new generation of fluorescent Ca2+ sensors excitable with a 488 nm laser, have been successfully used to image Ca2+ dynamics in the islet cell subpopulations39,40,41.
The new century brought forward two new trends that stemmed from neuroscience-related technological developments. Firstly, recombinant fluorescent sensors based on circular permutation of GFP variants substantially increased signal-to-noise ratio for Ca2+ detection, effectively bringing the studies to the level of large cell populations, in which the dynamics of [Ca2+]cyt in every cell could be resolved. Secondly, use of tissue-specific promoters allowed targeting sensor expression to minor subpopulations.
Although generally thought to reflect the developments in neuroscience, studies on islet Ca2+ dynamics have two key differences. Firstly, technologically, any in vivo imaging of islet signaling is more complex than imaging in the brain due to the unpredictable anatomy of the pancreas and the islets&#39; location42. Secondly, excellent electrical coupling between the islet &#946;-cells essentially renders islets into electrically inert populations displaying a seemingly perfect all-or-nothing response to the high glucose stimulus. We believe that studies of [Ca2+]i kinetics in minor islet subpopulations, such as &#948;-cells, based on tissue-specific targeting are likely to broaden our knowledge of their pharmacology/physiology. At the same time, highly sensitive probes allow expanding the statistical power of such measurements, accounting to islet-to-islet variability and allowing imaging of islets from different groups within one parallel experiment.

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.

<table><tbody><tr><td>40x/1.3 objective</td><td></td><td></td><td></td></tr><tr><td>Axiovert 200 microscope</td><td></td><td></td><td></td></tr><tr><td>emission</td><td></td><td></td><td></td></tr><tr><td>Excitation</td><td></td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma-Aldrich</td><td>F7524-500ML</td><td></td></tr><tr><td>Fluo4</td><td>Thermo Fisher (Life Technologies)</td><td>F14201</td><td></td></tr><tr><td>GCaMP6f, in (human type 5) adenoviral vector</td><td>Vector Biolabs</td><td>1910</td><td></td></tr><tr><td>Hanks&#039; solution</td><td>Thermo Fisher (GibCo, Life Technologies)</td><td></td><td></td></tr><tr><td>Liberase</td><td>Sigma-Aldrich</td><td>5401020001</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Thermo Fisher (GibCo, Life Technologies)</td><td>15140122</td><td></td></tr><tr><td>RPMI medium</td><td>Thermo Fisher (GibCo, Life Technologies)</td><td>61870044</td><td></td></tr><tr><td>Zeiss LSM510-META confocal system</td><td>Carl Zeiss</td><td></td><td></td></tr></tbody></table>

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 above, which provides exceptional stability and solution access.
Ca2+ spiking in &#945;-cells can be readily detected at low glucose levels (Figure 2). 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 (&#945;-cells?) at low glucose yet it has no effect on Ca2+ dynamics in most of the cells that are activated by low glucose (&#946;-cells).
When analyzed in terms of partial frequency (Figure 4A,C), adrenaline- or ghrelin-stimulated cells display a substantial increase under the all-or-nothing conditions. That is, a cell with low basal activity that gets activated by adrenaline or ghrelin exhibits a dramatic increase in this metric. However, the overall changes between basal spiking and the adrenaline effect are very subtle (Figure 4A,C). In contrast, partial AUC is sensitive to the changes introduced by adrenaline in all cells, even when basal activity is high (Figure 4B,D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59491/59491fig1.jpg" /> 
Figure 1: Loading of the trappable dye and expression of the recombinant sensor in islets. Typical mouse islets loaded with the trappable dye Fluo-4 (A) or expressing the recombinant sensor GCaMP6 in the peripheral layer of cells (B) or in the deeper layer (C). Polar tracer sulforhodamine B (SRB, shown as white) has been utilized to outline individual cells within each islet25. (D) Representative kinetics of Ca2+ in response to glucose recorded from individual cells within the islet using Fluo4. Note the heterogeneity within minor cell populations. <a href="https://www.jove.com/files/ftp_upload/59491/59491fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59491/59491fig2.jpg" /> 
Figure 2: Typical Ca2+ response of islet cells to various stimuli. Typical &#945;-cell (A) and &#948;-cell (B) Ca2+ dynamics, in response to adrenaline, glutamate, ghrelin, glucose. (C)-(D) Heat maps of the islet cell response showing adrenaline-positive (C) and ghrelin-positive (D) subpopulations. <a href="https://www.jove.com/files/ftp_upload/59491/59491fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59491/59491fig3.jpg" /> 
Figure 3: Baseline correction. <a href="https://www.jove.com/files/ftp_upload/59491/59491fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59491/59491fig4.jpg" /> 
Figure 4: Analysis of the time-lapse data. Analysis of the Ca2+ dynamics in &#945;-cells. Partial frequency (A), plateau fraction (B) and area under the curve (C) of an &#945;-cells [Ca2+]i trace. Populational [Ca2+]i data from a mouse pancreatic islet expressed as raw (F/F0) (D), partial frequency (E), plateau fraction (F) and area under the curve (G). <a href="https://www.jove.com/files/ftp_upload/59491/59491fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

imaging-calcium-dynamics-subpopulations-mouse-pancreatic-islet

Nanyang Technological University

Research

JoVE Journal

Biology

8.1K Views.  University of Oxford. Het protocol maakt een snelle geïsoleerde functie van kleine subpopulaties van cellen binnen pancreaseilandjes van Langerhans mogelijk. De voordelen van deze techniek omvatten de real-time aard en een hoog statistisch vermogen, en dus een hoge reproduceerbaarheid, van de verkregen gegevens. Om de eilandjes voor beeldvorming te immobiliseren, monteer je een beeldkamer voor een omgekeerde microscoop en plaats je een glazen afdekbrief in de kamer.Zorg ervoor dat de glazen kamerinterface ...

Video: Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

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, ...

Medicine

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 ...

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 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 ...

Immunology and Infection

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

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 ...

Imaging Calcium Dynamics in Pancreatic Cells: A Technique to Study Real-time Changes in Cytosolic Calcium Concentration in Pancreatic Islet

Source: Hamilton, A., et. al. <a href="https://www.jove.com/v/59491/" target="_blank">Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells</a>. J. Vis. Exp. (2019).
This video describes the technique of imaging islet cells in real-time, to study the changes in cytosolic calcium concentration in the minor subpopulations of the pancreatic islet cells. 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. This can provide an insight into the cross talk between the blood glucose and the islet secretory output.

Source: Hamilton, A., et. al. Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells. J. Vis. Exp. (2019).
This video describes the ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

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