The authors thank Marcia Boulina from the DRI imaging core facility for help with the microscopes. This work was supported by NIH grants 1K01DK111757-01 (JA), F31668418 (MM), R01 DK111538, R33 ES025673 and R56 DK084321 (AC).

The animal ethics committee of the University of Miami has approved all the experiments.
1. Viral Infection of Intact Isolated Human or Mouse Pancreatic Islets
<ol>
	<li>Islet culture: prepare the islet culture media: Connaught Medical Research Laboratories (CMRL) 1066, 10% (v/v) FBS and 2 mM L-Glutamine.
	<ol>
		<li>Human pancreatic islets are obtained from the Integrated Islet Distribution Program (NIDDK, NIH). Upon arrival, transfer the islets (~ 500 islet equivalents) to 35-mm non-tissue culture treated Petri dishes with 2 mL CMRL culture media at 37 &#176;C, 5%/95% CO2/O2 for 24 h before viral infection.</li>
		<li>Mouse pancreatic islets can be isolated following previously established protocols16. Following isolation, culture ~ 200 islet equivalents in 35-mm non-tissue culture treated Petri dishes with 2 mL CMRL culture media at 37 &#176;C, 5%/95% CO2/O2 for 24 h before viral infection. 
		NOTE: Avoid using transgenic mice with GFP or YFP reporters expressed on islets to avoid fluorescence overlap with NPY-pHluorin.</li>
	</ol>
	</li>
	<li>Virus preparation 
	NOTE: The NPY-pHluorin fusion was cloned into the pcDNA3 vector10 and subcloned into an adenoviral vector for adenoviral production [adenovirus serotype 5 (DE1/E3)] by a recombinant adenovirus manufacturing company. The virus was aliquoted and stored at -80 &#176;C. The viral stock is provided by the company at titers of 1012 to 1013 viral particles (~ 3 x 1010 - 3 x 1011 PFU).
	<ol>
		<li>For in vitro islet infection, use 106 PFU/mL, resulting in an approximate multiplicity of infection (MOI) of around 2 (see Discussion for details)</li>
	</ol>
	</li>
	<li>Viral infection of pancreatic islets 
	Caution: Working with adenoviruses requires Biosafety Level 2 (BSL2) procedures and certification. Check with the Institutional Biosafety Officer for guidance and training on BSL2 procedures.
	<ol>
		<li>Prepare human/mouse islets as described above.</li>
		<li>Add 5 - 10 &#181;L of stock virus to each 35-mm Petri dish containing human/mouse islets in 2 mL of CMRL culture media (with 10% FBS and 2 mM L-Glutamine). 
		NOTE: Adjust the volume of the virus used according to the viral titer as provided by the company datasheet.</li>
		<li>Culture the islets in virus-containing media at 37 &#176;C/5% CO2 for 24 h.</li>
		<li>After 24 h, aspirate the virus-containing media and replace with 2 mL CMRL culture media (with 10% FBS and 2 mM L-Glutamine).</li>
		<li>Culture the islets for 4 - 6 days at 37 &#176;C/5% CO2, replacing the media every 3 days.</li>
		<li>After 4 - 6 days of culturing, expect around 30% of islet cells to be infected. Islets can then be used for live imaging experiments.</li>
	</ol>
	</li>
</ol>
2. Confocal Imaging of Infected Islets
NOTE: Refer to the Table of Materials for the materials and equipment required for confocal imaging.
<ol>
	<li>Reagent preparation and experimental setup
	<ol>
		<li>Prepare extracellular solution: add 125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl2, 1 mM MgCl2, 25 mM HEPES, 0.1% BSA, pH 7.4, sterile filtered. 
		NOTE: This buffer is normally prepared without glucose and can be stored at 4 &#176;C for up to 1 month. Glucose is added on the day of the experiment to reach the desired final concentration.
		<ol>
			<li>Prepare basal glucose (3 mM) medium: add 75 &#181;L of 2 M glucose stock to 50 mL of extracellular solution.</li>
			<li>Hyperglycemic (16 mM) medium: add 400 &#181;L of 2 M glucose stock to 50 mL of extracellular solution</li>
		</ol>
		</li>
		<li>Dilute any additional stimulus (e.g., KCl or adenosine triphosphate (ATP)) in extracellular solution containing 3 mM glucose.</li>
		<li>Before starting an experiment, pretreat the coverslips with poly-D-lysine by adding 30 &#181;L of poly-D-Lysine solution (1 mg/mL) to the coverslip for 1 h and thoroughly rinsing it with H2O. 
		NOTE: The poly-lysine coated coverslips can be stored at room temperature for up to 6 months.</li>
		<li>At least 1 h before the experiment, using a 1 mL pipette, transfer the islets to a 35-mm Petri dish containing extracellular solution with 3 mM glucose. Keep the islets at 37 &#176;C and 5% CO2. 
		NOTE: If needed, the plasma membrane can be labeled in this step. To label the plasma membrane, add 2 &#181;M di-8-ANEPP dye to the extracellular solution with 3 mM glucose. Incubate the islets in dye solution for 1 h at 37 &#176;C/5% CO2. The plasma membrane dye can be excited at 488 nm and detected at 620 nm.</li>
		<li>min before starting an experiment, attach the coverslip to the imaging chamber by sealing it with vacuum silicone grease. Fix the imaging chamber to the imaging platform. Using a pipette, place 20 - 30 islets on the poly-D-Lysine-treated area of the coverslip and let the islets adhere to the surface for 20 min. 
		NOTE: It is important not to let the coverslip dry completely to avoid islet damage.</li>
		<li>While the islets are adhering to the coverslip, prepare the perfusion system by thoroughly rinsing it with water. Add each solution to a different channel: 3 mM glucose (channel 1), 16 mM glucose (channel 2), 16 mM glucose with 100 &#181;M 3-isobutyl-1-methylxanthine (IBMX) and 10 &#181;M forskolin (channel 3), 25 mM KCl in 3 mM glucose (channel 4), 10 &#181;M ATP in 3 mM glucose (channel 5). Remove all the bubbles from the system by opening each channel separately and letting the solution flow for a few minutes, and make sure that the flow is consistent (0.5 mL/min) and the tubing is not leaking.</li>
		<li>Connect the single inline solution heater to the perfusion outlet tube and adjust the temperature of the outflowing buffer to 37 &#176;C.</li>
		<li>Prepare the suction pump. Remove all the bubbles from the system, and make sure that the flow is consistent and the tubing is not leaking.</li>
		<li>Once the islets adhere to the coverslip surface, gently fill up the imaging chamber with the extracellular solution containing 3 mM glucose. Avoid washing the islets away from the surface of the coverslip.</li>
		<li>Place the imaging platform with the islets onto the microscope stage and connect it to the perfusion system and suction pump.</li>
		<li>Turn on the flow and constantly perfuse the islets with the 3G extracellular solution. The system is now ready for confocal imaging.</li>
	</ol>
	</li>
	<li>Confocal imaging
	<ol>
		<li>Locate the islets in the microscope field with lower magnification. Once focused on the islets, switch to higher magnification objectives (e.g., 63X water immersion objective (63X/0.9 NA)).</li>
		<li>Open the acquisition using software (Table of Materials) and activate the resonant scanner mode.</li>
		<li>Select the XYZT imaging mode and configure the acquisition settings as follows:
		<ol>
			<li>Turn on the Argon laser and the 488 nm laser line and adjust the laser power to 50% for pHluorin excitation.</li>
			<li>Collect emission at 505 - 555 nm.</li>
			<li>Choose a resolution of 512 x 512 pixels. Press the &#34;Live&#34; button to start imaging and adjust the gain levels (typical gain is around 600 V).</li>
			<li>Set the begin and end of the z-stack: focus on the top of the islet and choose &#34;begin&#34; and move to the last plane that can be focused and choose &#34;end&#34;. Use a z-step size of 5 &#181;m. The software will automatically calculate the number of confocal planes.</li>
			<li>Set the time interval for acquisition of each z-stack close to 1.5 - 2 s and choose the option &#34;Acquire until stopped&#34; for continuous imaging.</li>
			<li>Press the &#34;start&#34; button to initialize.</li>
		</ol>
		</li>
		<li>Use various stimulation protocols to induce granule exocytosis by perfusing the islets with the desired stimuli. Stimulation protocols can be customized to fit the desired scientific purpose (see below).</li>
	</ol>
	</li>
	<li>Stimulation protocols 
	NOTE: In every stimulation protocol, start by recording at least 2 min of islet background activity during constant perfusion with extracellular solution containing 3 mM glucose. Perfuse with a stimulant for the desired period of time. The order of stimulants, duration of stimulation as well as duration of recordings can be customized to fit the desired scientific purposes. Make sure to wash islets thoroughly with extracellular solution containing 3 mM glucose before starting a new stimulation. Below find the sample stimulation protocols that have been used to demonstrate the method capabilities.
	<ol>
		<li>Stimulate with ammonium chloride (NH4Cl) as a positive control for pHluorin pH sensitivity and viral infection efficiency (Figure 3): 3 mM glucose (2 min) &#8594; 50 mM NH4Cl (2 min) in 3 mM glucose&#160;&#8594; 3 mM glucose (2 min) 
		NOTE: In the NH4Cl solution, replace NaCl on an equimolar basis.</li>
		<li>Stimulate insulin granule exocytosis by increasing the glucose concentration (Figure 5 and Figure 6): 3 mM glucose (2 min)&#160;&#8594; 16 mM glucose (15-30 min)&#160;&#8594; 3 mM glucose (2 min 
		NOTE: In order to see several bursts of activity, perfuse the islets continuously with the 16G solution for at least 15 min. 
		NOTE: In order to increase the consistency of secretory responses17, users may add cAMP-raising agents (100 &#181;M IBMX and 10 &#181;M forskolin) to both the 3G and 16G solutions. This does not change the temporal patterns of granule secretion. For details see15 and Discussion.</li>
	</ol>
	</li>
</ol>

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The authors declare they have no competing financial interests.

This manuscript describes a technique that can be used to visualize exocytosis of insulin granules in beta cells within intact pancreatic islets by confocal microscopy. It uses NPY-pHluorin as the fluorescent reporter cloned into adenovirus to ensure a high transfection efficiency.
Although the method was highly efficient in our hands, it might require some modifications that primarily depend on two parameters: 1) the quality of islet preparation, and 2) the titer of viral stock with optimization of infection conditions. Prior to the viral infection of islets, allow the islet preparation to recover from isolation and transportation stress overnight at 37 &#176;C. Inspect the islet morphology under a light microscope throughout the infection/expression period and check for signs of decreased viability such as cells protruding from the islet surface or presence of hypoxic cells (darker cells) in the islet center18. Each viral stock may vary in titer and infection efficiency. Make sure to adjust the volume of the virus used according to the viral titer and the time of culture prior to the microscopy assay. The most critical step is the optimization of the infection conditions (e.g., number of viral particles, culture time before assay) so that enough of the reporter is expressed and the islet integrity and responsiveness are maintained. When optimizing the infection protocol and with each new viral batch, collect a few islets at different time points and check whether exocytosis can be elicited with KCl (30 mM) and/or high glucose (16 mM). We found that the assay works better and islet cells remain healthy and glucose responsive when the MOI is low (around 2) and the culture time is extended (around 5 days) to increase the expression levels of the fusion construct. Our results are in line with a previous study showing that efficient expression of gene products can be better achieved at lower transfecting concentrations of the adenoviral vector while preserving islet function19.
The technique has, however, some limitations. One is that by using NPY-pHluorin as a reporter, granules cannot be visualized before they fuse with the plasma membrane and the fusion pore opens. To study insulin granule trafficking instead of exocytosis, adenovirus encoding NPY-eGFP can be used instead. Another limitation is that, to a lower extent, NPY-pHluorin can be targeted to non-insulin containing granules. However, the majority of the cells that were infected with NPY-pHluorin also expressed insulin and less than 10% of the cells expressed glucagon or somatostatin. Indeed, the glucose dependency of granule secretion, its pulsatile pattern and co-localization with insulin, indicated that most secretory events represent cargo release from insulin granules. To guarantee monitoring of insulin exocytosis is occurring, the assay should be performed under conditions that stimulate insulin secretion (e.g., high glucose). Nevertheless, to improve the specificity of beta cell expression, NPY-pHluorin could be put under the control of a more selective promoter such as the rat insulin promoter. Another limitation of the technique is that it requires expression of exogenous proteins in insulin granules, which can perturb their biogenesis and/or behavior20. This should be considered when interpreting the results of the experiments and, if possible, the conclusions should be validated using other insulin granule cargo proteins (e.g., C-peptide, islet amyloid polypeptide) as reporters.
This protocol also recommends including cAMP-raising agents (IBMX and forskolin) to potentiate the effects of high glucose on insulin granule exocytosis. This stimulation protocol mimics the activation of physiologically relevant amplifying pathways (triggered by incretins or paracrine and neural signaling molecules) that potentiate insulin secretion by increasing cAMP in the pancreatic beta cells. Nevertheless, in the absence of cAMP raising agents, high glucose (16 mM) also elicits granule exocytosis but the number of secretory events observed is lower. Granules still appear in discrete bursts, are synchronized with the average cell response, and show similar burst periods: 1.4 - 6.6 min in high glucose plus IBMX/forskolin vs. 1.5 - 10 min in high glucose alone.
The assay described here is significant with respect to existing methods as it has sufficient spatial and temporal resolution to visualize single fusion events in real time in beta cells within intact islets. For instance, it revealed the high synchronization with which insulin granules are released in human islets upon glucose stimulation. Moreover, if islets are well attached to the coverslip, they can be imaged for prolonged periods (at least 15 - 20 min). This was used to show that in human islets exocytosis occurs in distinct bursts that appear in periods similar to those of pulsatile in vivo insulin secretion. The assay also allows for testing the effect of several stimuli on insulin exocytosis using murine or human islets. Furthermore, because it does not require islet cell dissociation like other methods, it can be applied to study the behavior of beta cell populations within an islet. Indeed, we observed that neighboring beta cells form clusters of ~ 5 - 10 cells in which activity is synchronized. Beta cells from the same cluster are probably coupled by gap junction channels made of connexin 3621. Their synchronized response illustrates the beta cell connectivity that is essential for proper insulin secretion22.
In the future, this technique can be combined with other fluorescent dyes for functional imaging to visualize in real time how changes in cytosolic calcium or membrane potential, for instance, correlate with granule exocytosis in beta cells. In particular, as new pH sensitive red fluorescent proteins are becoming available14,23 and can be fused to NPY or other insulin granule cargo proteins, green dyes for functional imaging can be used. Furthermore, this technique can be combined with a mouse model for in vivo imaging of vascularized islets transplanted into the anterior chamber of the mouse eye24. Islets can be infected with adenoviruses 48 - 72 h before transplantation into the eye. Immune deficient mice should be used if human islets are transplanted. Using this model, the subcellular spatial pattern of insulin granule secretion can be correlated with vascular arrangements25.

Insulin is produced by the beta cells of the pancreatic islet and it is a key regulator of glucose metabolism1. Death or dysfunction of beta cells disturbs glucose homeostasis and leads to diabetes2. Insulin is packed in dense-core granules that are released in a Ca2+-dependent manner3. Elucidating how insulin granule exocytosis is regulated is essential to fully understand what determines insulin secretion and opens new avenues for the identification of novel therapeutic targets for the treatment of diabetes.
Insulin exocytosis has been extensively studied using electrophysiological approaches, such as membrane capacitance measurements, and microscopic approaches in combination with fluorescent molecules. Membrane capacitance measurements have good temporal resolution and allow single cell recordings. However, changes in the capacitance reflect the net surface change of the cell and do not capture individual fusion events or distinguish insulin granule fusion from other non-insulin secretory vesicles3. Microscopic approaches, such as two-photon or total internal reflection fluorescence (TIRF) microscopy in combination with fluorescent probes and vesicle cargo proteins, provide additional detail. These techniques capture single exocytotic events and also the pre- and post-exocytotic stages and can be used for studying exocytotic patterns in populations of cells3.
Fluorescent reporters can be of three types: 1) extracellular, 2) cytoplasmic, or 3) vesicular. 1) Extracellular reporters are polar tracers (e.g., dextrans, sulforhodamine B (SRB), lucifer yellow, pyranine) that can be introduced through the extracellular milieu4. The use of polar tracers allows for the investigation of the fusion pore in a population of cells and captures various intercellular structures such as blood vessels. However, they do not report on vesicle cargo behavior. 2) Cytoplasmic reporters are fluorescent probes coupled to membrane-associated proteins that face the cytoplasm and are involved in docking and exocytosis. Examples include members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family that have been successfully used in neuroscience for studying neurotransmitter release5. Such proteins have multiple binding partners and are not insulin-granule specific. 3) Vesicular reporters are fluorescent probes fused to vesicular cargo proteins that allow for the investigation of cargo-specific vesicle behavior. Insulin-granule specific cargo proteins include insulin, c-peptide, islet amyloid polypeptide, and NPY among others6,7. NPY is only present in insulin containing granules, and is co-released with insulin, making it an excellent partner for a fluorescent reporter8.
The fusion of different fluorescent proteins to NPY has been previously employed to study various aspects of exocytosis in neuroendocrine cells, such as the requirement of specific synaptotagmin isoforms9,10 and how the time-course of release depends on the actin cytoskeleton and on myosin II11,12. In this study, we chose pHluorin as the fluorescent reporter, which is a modified GFP that is non-fluorescent at the acidic pH inside dense core granules but becomes brightly fluorescent upon exposure to the neutral extracellular pH13. Mature insulin granules have an acidic pH below 5.5. Once the granule fuses with the plasma membrane and opens, its cargo is exposed to the neutral extracellular pH of 7.4, allowing the use of the pH-sensitive proteins pHluorin as a reporter7,14.
In view of the pH sensitive nature of pHluorin and the selective expression of NPY in insulin granules, the NPY-pHluorin fusion construct can be used to study various properties of insulin granule exocytosis. The viral delivery of the fusion construct ensures high transfection efficiency and works on primary beta cells or cell lines as well as on isolated islets. This method can also be used as a guideline for studying exocytosis in any other cell type with NPY-containing vesicles. It can also be combined with any transgenic mouse model to study effects of certain conditions (knockdowns, overexpression, etc.) on exocytosis. This technique has been previously used to characterize spatial and temporal patterns of insulin granule secretion in beta cell populations in human islets15.

<table><tbody><tr><td>Upright laser-scanning confocal microscope</td><td>Leica Microsystems, Wetzlar, Germany</td><td>TCS-SP5</td><td>includes LAS AF, the image acquisition software</td></tr><tr><td>Imaging chamber</td><td>Warner instruments</td><td>RC-26</td><td></td></tr><tr><td>Imaging chamber platform</td><td>Warner instruments</td><td>PH-1</td><td></td></tr><tr><td>22 x 40 glass coverslips</td><td>Daiggerbrand</td><td>G15972H</td><td></td></tr><tr><td>Vacuum silicone grease</td><td>Sigma</td><td>Z273554-1EA</td><td></td></tr><tr><td>Multichannel perfusion system</td><td>Warner instruments</td><td>VC-8</td><td></td></tr><tr><td>Single inline solution heater</td><td>Warner instruments</td><td>SH-27B</td><td></td></tr><tr><td>Temperature controller</td><td>Warner instruments</td><td>TC-324C</td><td></td></tr><tr><td>Peristaltic Suction pump</td><td>Pharmacia</td><td>P-1</td><td></td></tr><tr><td>35 mm Petri dish, non-tissue culture treated</td><td>VWR</td><td>10861-586</td><td></td></tr><tr><td>CMRL Medium, no glutamine</td><td>ThermoFisher</td><td>11530037</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>ThermoFisher</td><td>16140071</td><td></td></tr><tr><td>L-Glutamine 200 mM</td><td>ThermoFisher</td><td>25030081</td><td></td></tr><tr><td>5 M NaCl solution</td><td>Sigma</td><td>S5150</td><td></td></tr><tr><td>3 M KCl solution</td><td>Sigma</td><td>60135</td><td></td></tr><tr><td>1 M CaCl&lt;sub&gt;2 &lt;/sub&gt;solution</td><td>Sigma</td><td>21115</td><td></td></tr><tr><td>1 M MgCl&lt;sub&gt;2 &lt;/sub&gt;solution</td><td>Sigma</td><td>M1028</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>1 M HEPES solution</td><td>Sigma</td><td>H0887</td><td></td></tr><tr><td>Vacuum filter</td><td>VWR</td><td>431098</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma-aldrich</td><td>P6407</td><td></td></tr><tr><td>Di-8-ANNEP</td><td>ThermoFisher</td><td>D3167</td><td></td></tr><tr><td>3-isobutyl-1-methylxanthine (IBMX)</td><td>Sigma</td><td>I5879</td><td></td></tr><tr><td>Forskolin</td><td>Sigma</td><td>F3917</td><td></td></tr></tbody></table>

confocal imaging of neuropeptide y-phluorin: a technique to visualize insulin granule exocytosis in intact murine and human islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

The entire workflow of the technique is shown in Figure 1. Briefly, mouse or human islets can be infected with adenovirus encoding NPY-pHluorin and imaged, after few days in culture, under a confocal microscope. As granules fuse with plasma membrane and open, an increase in fluorescence is observed and can be quantified (Figure 1). To determine if NPY-pHluorin is indeed a suitable tool to monitor insulin granule dynamics, infected islets were immunostained with antibodies against GFP and the different islet hormones insulin, somatostatin or glucagon. Most cells expressing NPY-pHluorin (GFP positive) were beta cells as they also expressed insulin (~ 90%; Figure 2A, 2B). Only a few glucagon-positive alpha cells or somatostatin-positive delta cells were GFP-labeled (Figure 2B). Importantly, in infected cells, the NPY fusion colocalized with insulin (Pearson&#39;s correlation coefficient of 0.61 &#177; 0.04 for GFP and insulin vs. 0.21 &#177; 0.05 for GFP and glucagon and 0.07 &#177; 0.01 for GFP and somatostatin).
We demonstrated that pHluorin is an effective pH sensor, as increasing the intracellular pH with 50 mM NH4Cl resulted in a &#62;500% increase in the intracellular fluorescence (Figure 3A, 3B, Video 1). As the pH inside the granule increases upon fusion with the plasma membrane and the opening of the fusion pore, granules containing NPY-pHluorin become visible upon conditions that stimulate insulin exocytosis such as membrane depolarization with KCl (Figure 4, Video 2).
Single secretory events in beta cells within intact islets can be visualized with confocal time-lapse imaging of NPY-pHluorin infected islets. These occur in response to direct membrane depolarization with KCl or several other physiological stimuli (Figure 4 and Figure 7, see below). In the basal extracellular glucose concentration (3 mM), little secretory activity is observed. However, granule fusion with the plasma membrane is triggered in response to stimulation with high glucose (16 mM) (Figure 5A, Video 3). By placing the ROIs with different sizes and in different areas, the response kinetics of secretory granules can be compared with that of individual cells or groups of cells in close proximity (cluster) (Figure 5B). This type of analysis enabled determining that: 1) the individual secretory events are very synchronized and secreted within a few seconds from the average cell response, and 2) the beta cells in close proximity form functional clusters of synchronized activity.
Insulin secretion in the body occurs in a pulsatile manner. Stimulating the NPY-pHluorin-infected islets with elevated glucose concentration for prolonged periods (15 - 20 min) gave rise to multiple pulses (or bursts) of secretion (Figure 6, Video 4). During each pulse, individual granules fuse with the plasma membrane in a synchronized fashion as shown before (Figure 5). Bursts of secretory activity can be seen every 3 - 4 min.
Transient increases in fluorescence in NPY-pHluorin containing granules could also be observed in response to the purinergic agonist ATP (10 &#181;M) or the muscarinic agonist acetylcholine (ACh, 10 &#181;M) (Figure 7), both of which are known stimulators of insulin secretion in human islets. As expected, the membrane depolarization with KCl (30 mM) also triggered insulin granule exocytosis (Figure 5). Several stimuli can be applied to the same islet preparation as long as the islets are washed well in between stimuli. Make sure to alter the order of application when repeating the experiment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56089/56089fig1.jpg" /> 
Figure 1. A Scheme Illustrating the Assay Developed. Adenovirus that encodes a pH-sensitive GFP (pHluorin) coupled to NPY were produced and used to infect mouse or human pancreatic islets. Four to six days after infection, the islets were placed on a coverslip and imaged under an upright laser-scanning confocal microscope. The NPY-pHluorin construct is expressed in insulin granules in beta cells. Its fluorescence is quenched at the acidic pH of mature insulin granules but becomes bright upon granule fusion with the plasma membrane and exposure to the neutral extracellular pH of 7.4.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56089/56089fig2.jpg" /> 
Figure 2. The NPY Fusion Construct is Expressed in Insulin Granules. (A) Confocal images of intact human islets infected with NPY-pHluorin, immunostained for GFP (green) and insulin (left panel, red), somatostatin (middle, red) or glucagon (right, red). The cell nuclei are shown in blue (DAPI labeled). Scale bars = 10 &#181;m. (B) Quantification of the fraction of GFP-positive cells that also contain either insulin (Ins), somatostatin (Soma) or glucagon (Glu). Shown are the mean &#177; SEMs, n = 5 islets. <a href="https://cloudflare.jove.com/files/ftp_upload/56089/56089fig2large.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/56089/56089fig3.jpg" /> 
Figure 3. NPY-pHluorin is an Effective pH Sensor. (A) Maximal projection of confocal images of a mouse islet infected with NPY-pHluorin in extracellular solution containing 3 mM glucose before (upper panel, 3G) and in the presence of 50 mM NH4Cl (lower panel). Scale bar = 100 &#181;m. (B) Trace showing the change in mean fluorescence intensity (arbitrary units) in the whole islet induced by NH4Cl. Dashed line indicates NH4Cl application. <a href="https://cloudflare.jove.com/files/ftp_upload/56089/56089fig3large.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/56089/56089fig4.jpg" /> 
Figure 4. Granules Containing NPY-pHluorin Become Visible Once They Fuse with the Plasma Membrane and Open. Human isletsinfected with NPY-pHluorin adenovirus were incubated with the plasma membrane dye di-8-ANEPP (red). Islet stimulation with KCl (30 mM) triggered a sudden and transient appearance of fluorescent granules at the cell surface (shown in green). Confocal images of cells within a human islet are shown at before the exocytotic response (t = 0), during (t =3 - 9 s) and after (t = 12 s). Contrast was adjusted to remove background green fluorescence. Scale bar = 20 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56089/56089fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56089/56089fig5.jpg" /> 
Figure 5. High Glucose Triggers NPY-pHluorin Fusion with the Plasma Membrane. (A) Maximal projection of confocal images of an intact human islet infected with NPY-pHluorin in basal glucose concentration (3G - 3 mM Glucose, left panel) or in high glucose (16G - 16 mM Glucose, right panel). Extracellular solutions contained the cAMP raising agents forskolin and IBMX (see Protocol for details). Scale bar = 10 &#181;m. (B) Traces showing changes in mean fluorescence intensities (arbitrary units) in the green channel in ROIs placed around single secretory events (red, granule), cells (green) or cell clusters (blue). High glucose was applied for 5 min after ~ 2.5 min in 3G. Fluorescence values were normalized to the initial fluorescence value (baseline). <a href="https://cloudflare.jove.com/files/ftp_upload/56089/56089fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56089/56089fig6.jpg" /> 
Figure 6. Secretory Events Generate Discrete Pulses of Secretion upon High Glucose. Traces showing changes in mean fluorescence intensity (arbitrary units) in ROIs placed around different cells within an intact human islet during sustained stimulation with 16 mM glucose. Each color represents an individual cell. Burst of secretory activity can be seen every 3 - 4 min. <a href="https://cloudflare.jove.com/files/ftp_upload/56089/56089fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56089/56089fig7.jpg" /> 
Figure 7. NPY-pHluorin Fusion with the Plasma Membrane is Triggered by Known Stimulators of Insulin Secretion. Traces showing changes in mean fluorescence intensity (arbitrary units) in ROIs placed around individual secretory events in cells within intact human islets induced by KCl (30 mM), ATP (10 &#181;M), high glucose (16G, 16 mM) and acetylcholine (Ach, 10 &#181;M). KCl and ATP were applied in the basal glucose concentration (3 mM). Black lines show the average granule and gray lines reflect SEM values. Green traces show the corresponding cell response. Horizontal time scale (2 min) applies to all graphs. <a href="https://cloudflare.jove.com/files/ftp_upload/56089/56089fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/56089/56089video1.jpg" /> 
Video 1. NPY-pHluorin Adenovirus Efficiently Infect Islet Cells and Sense pH Changes. 
Mouse islets were infected for 4 days with adenovirus encoding NPY-pHluorin. Infected islets were placed on a coverslip and mounted on the imaging chamber. To increase intracellular pH, the islets were perfused with 50 mM NH4Cl added to the extracellular solution containing 3 mM glucose. A fast and strong increase in fluorescence can be seen upon NH4Cl application. Scale bar = 100 &#181;m. Movie speed = 5 fps. Total duration of the movie is 90 s. <a href="http://cloudflare.jove.com/files/ftp_upload/56089/Video_1_JoVe.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 2" src="/files/ftp_upload/56089/56089video2.jpg" /> 
Video 2. Granules Containing NPY-pHluorin Become Visible Once They Fuse with the Plasma Membrane and Open. 
Human islets infected with NPY-pHluorin adenovirus were incubated with the plasma membrane dye di-8-ANEPP. Islet stimulation with KCl (30 mM) triggered a sudden and transient appearance of fluorescent granules at the cell surface. A maximal projection of confocal planes is shown. Contrast was adjusted to remove background green fluorescence. Scale bar = 20 &#181;m. Movie speed = 5 fps. Total duration of the movie is 60 s. <a href="http://cloudflare.jove.com/files/ftp_upload/56089/Video_2_JoVe.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 3" src="/files/ftp_upload/56089/56089video3.jpg" /> 
Video 3. High Glucose Triggers Exocytosis of Granules Containing NPY-pHluorin. 
Fusion of NPY-pHluorin containing granules is triggered in response to stimulation with high glucose (16 mM). In basal extracellular glucose concentration (3 mM), little secretory activity is observed. However, upon high glucose, insulin granules transiently appear at the plasma membrane of different cells in an islet in a synchronized fashion. High glucose was applied after ~ 2.5 min in 3G (16G label appears). A maximal projection of confocal planes is shown. Scale bar = 20 &#181;m. Movie speed = 10 fps. Total duration of the movie is 7 min. <a href="http://cloudflare.jove.com/files/ftp_upload/56089/Video_3_JoVe.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 4" src="/files/ftp_upload/56089/56089video4.jpg" /> 
Video 4. Prolonged Stimulation with High Glucose Triggers Several Bursts of Exocytosis. 
Prolonged stimulation with high glucose gives rise to multiple pulses of secretion islets, during which granules transiently appear. Pulses of secretory activity can be seen every ~ 3 - 4 min. Confocal plane of a human islet is shown. A pseudocolor scheme was applied for fluorescence intensity. Scale bar = 20 &#181;m. Movie speed = 30 fps. Total duration of the movie is 20 min. <a href="http://cloudflare.jove.com/files/ftp_upload/56089/Video_4_JoVe.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets at JoVE.com

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

confocal-imaging-neuropeptide-y-phluorin-technique-to-visualize

Nanyang Technological University

Research

JoVE Journal

Biology

9.1K Views.  University of Miami. We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Video: Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

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

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

Bioengineering

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

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 ...

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 ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

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

Intracellular Signaling Events and Hormone Secretion in Human Pseudoislets

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Author Spotlight: Investigating Islet Abnormalities and Function with a Pseudoislet Protocol 

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, ...

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 ...

Isolated Pancreatic Islet Treatment and Apoptosis Measurement

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing &#946;-cells, using a combination of fluorescence staining techniques and confocal microscopy to assess cell viability, apoptosis, and &#946;-cell-specific death. Isolated mouse islets were treated with varying concentrations of a cytokine cocktail, including TNF-&#945;, IL-1&#946;, and IFN-&#947;, to mimic immune-mediated apoptosis during the development of type 1 diabetes. The viability of islet cells was evaluated with FDA/PI dual staining, where FDA conversion to fluorescein indicated viable cells, and PI marked membrane-compromised cells. YOPRO-1 and nuclear staining provided additional data on apoptosis, with Annexin-V confirming early apoptotic cells. Quantitative analysis revealed significant increases in apoptosis and cell death rates in cytokine-treated islets. To specifically assess effects on &#946;-cells, Zn2+ selective indicator staining was used to label insulin-producing cells through the zinc association in insulin granules, revealing substantial &#946;-cell loss following treatment of islets with pro-inflammatory cytokines for 24 h. These multi-staining protocols effectively capture and quantify the extent of cytokine-induced damage in islets and can be used to evaluate therapeutics designed to prevent &#946;-cell apoptosis in early type 1 diabetes.

This study utilizes multi-staining fluorescence-based markers of cell death and apoptosis combined with confocal microscopy to assess cytokine-induced apoptosis and &#946;-cell-specific death in pancreatic islets. It reveals spatial and temporal changes in cell death and apoptosis in response to extracellular stimuli.

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing &#946;-cells, using a combination of ...

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 ...