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 optimizat...

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 width="845">
	<tbody>
		<tr height="63">
			<td height="63" style="width:280px;height:63px;">Upright laser-scanning confocal microscope</td>
			<td style="width:232px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:167px;">TCS-SP5</td>
			<td style="width:167px;">includes LAS AF, the image acquisition software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">RC-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber platform</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">PH-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">22x40 glass coverslips</td>
			<td style="width:232px;">Daiggerbrand</td>
			<td style="width:167px;">G15972H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum silicone grease</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">Z273554-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Multichannel perfusion system</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">VC-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Single inline solution heater</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">SH-27B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Temperature controller</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">TC-324C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Peristaltic Suction pump</td>
			<td style="width:232px;">Pharmacia</td>
			<td style="width:167px;">P-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:280px;height:42px;">35 mm Petri dish, non-tissue culture treated</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">10861-586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">CMRL Medium, no glutamine</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">11530037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">FBS, heat inactivated</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">16140071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">L-Glutamine 200mM</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">5M NaCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">S5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3M KCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">60135</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M CaCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">21115</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M MgCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">M1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Bovine Serum Albumin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">1M HEPES solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum filter</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">431098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">D-Glucose</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Poly-D-lysine hydrobromide</td>
			<td style="width:232px;">Sigma-aldrich</td>
			<td style="width:167px;">P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Di-8-ANNEP</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">D3167</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3-isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">I5879</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Forskolin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">F3917</td>
			<td style="width:167px;"></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, infecte...

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 ...

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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

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

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

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

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

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

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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

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

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

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

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...