Name: Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets (Video) | JoVE Journal
Uploaded: 2015-05-11T14:00:00.000+00:00
Duration: 10 min 9 s
Description: Learn pancreatic islet isolation from Lepr db mice and live-cell two-photon imaging to assess insulin secretion dynamics in type 2 diabetes research. This protocol enables real-time visualization of beta cell granule fusion events.

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5 minutes

This work was supported by an Australian Research Council Grant DP110100642 (to PT) and National Health and Medical Research Council Grants APP1002520 and APP1059426 (to PT and HYG).

NOTE: All present experiments were performed according to local animal ethics procedures of the University of Queensland (approved by the University of Queensland, Anatomical Biosciences Ethics Committee).
1. Islet Isolation
<ol>
	<li>Reagent preparation
	<ol>
		<li>Mixture of enzymes
		<ol>
			<li>For the pancreatic digestion, use a mixture of liberase and collagenase type IV. Dilute liberase TL (thermolysin low) 1 vial of 5 mg with 26 ml of DMEM (Dulbecco&#8217;s Modified Eagle Medium).</li>
			<li>Prepare collagenase type IV in Hank&#8217;s buffer at a concentration of 0.5mg/ml, supplemented with 5mM HEPES, 0.5mM CaCl2, 0.1mg/ml DNAse and 1mg/ml bovine serum albumin.</li>
			<li>Mix the liberase TL and collagenase solutions in a ratio of 4:1 (by volume), i.e., 4 ml liberase TL + 1 ml collagenase. Aliquot the mixture of enzymes to 2.5ml each and store at -20 &#176;C. 
			NOTE: The tissue incubation times vary a bit between batches of enzyme (30 sec&#160;to 1 min&#160;difference). Therefore, a batch test may be required.</li>
		</ol>
		</li>
		<li>RPMI Isolation Media
		<ol>
			<li>Dissolve one vial of RPMI 1640 powder in 1 L of water at RT and supplement with 2 g NaHCO3, 4.02 g HEPES.</li>
			<li>Combine the RPMI isolation media in a large conical flask, mix with magnetic stirrer, adjust to pH 7.4 with NaOH, filter sterilize and store at 4 &#176;C.</li>
		</ol>
		</li>
		<li>RPMI Culture Media</li>
		<li>Use RPMI culture media, with 10% fetal bovine serum and 1% antibiotics (i.e., penicillin 100 U/ml, streptomycin 100 &#956;g/ml).</li>
	</ol>
	</li>
	<li>Islet Isolation and Culturing
	<ol>
		<li>Animal Sacrifice
		<ol>
			<li>Sacrifice mice by cervical dislocation or CO2 asphyxiation according to the local institution&#8217;s ethics procedures. Spray the mouse body thoroughly with 70% ethanol.</li>
		</ol>
		</li>
		<li>Pancreatic Perfusion
		<ol>
			<li>Use 2 long cuts laterally through the abdominal skin and peritoneum to make a V-shaped flap. Pull the flap of the skin up to uncover the entire abdominal cavity, put aside the bowel and keep the liver in place by a folded tissue paper to reveal the common bile duct (Figures&#160;1 and 2). Put the animal so that the head toward the surgeon and the tail away from the surgeon.</li>
			<li>Apply a vascular clamp at the ampulla on the duodenum wall to block the injected enzymes from entering the duodenum (Figure&#160;1). 
			NOTE: This is an important step as it determines whether the injected enzymes will go into the pancreatic duct and perfuse the pancreas or reflux to the injection site (over clamp) or go into the duodenum (under clamp).</li>
			<li>Cannulate the common bile duct with a 31 G needle near the junction of the common hepatic duct and cystic duct (Figure&#160;1) and leave room for a second injection if the pancreas fails to perfuse the first time. Slowly (over ~10 sec) inject about 2 ml of cold enzyme mixture into the mouse common bile duct. 
			NOTE: The pancreas will be perfused quickly if the clamp is in the right position.</li>
			<li>Adjust the clamp position if the needle is inside the common bile duct but cannot perfuse the pancreas (i.e., move up the clamp a bit away from the liver if over clamped or move it down a bit to the liver if under clamped). 
			NOTE: The needle sometimes goes into the surrounding capsule rather than the lumen of the duct. A useful sign to check whether the needle is inside the common bile duct is the dilation of the duct when injecting. If the pancreas fails to perfuse, an alternative is the enzyme mixture could be injected to multiple sites of the pancreas with lower yield expected. The perfusion of the left site of the pancreas (the splenic lobe) is important for maximization of the islet yield.</li>
			<li>After the pancreas is perfused, quickly remove the pancreas, put it into a vial (~20 ml, glass) and incubate for ~19 min&#160;in a 37 &#176;C water bath. 
			NOTE: The incubation time can vary a bit depending on the mouse strain and age.</li>
			<li>After the incubation time, stop the digestion process by adding ~20 ml of cold isolation media. Shake the vial gently to disrupt the pancreas and pour it through a normal tea sieve (pore diameter of 1 mm) into a sterile 50 ml tube. Top up the tube with isolation media and centrifuge at 400 x g for 1 min&#160;at 4 &#176;C with brake off.</li>
			<li>Dissolve the pellet in the isolation media again and transfer to two 15 ml tubes, then centrifuge at 400 x g for 1 min&#160;at 4&#176;C with brake off. After aspiration the supernatant, mix the two pellets well with 6 ml of density gradient cell separation media (Histopaque 1077 solution) per tube by vortexing or pipetting up and down. Then gently add ~6 ml of the isolation media to the side of the tube on top of the density gradient separation media and centrifuge at 100 x g for 15 min&#160;at 4 &#176;C with brake off.</li>
			<li>Collect the supernatant in the upper (isolation media), middle and lower (density gradient cell separation media) layer in 3 separate dishes. Handpick the islets in these 3 dishes, using a pipette under a dissecting microscope. 
			NOTE: The middle layer contains most of the islets. Islets are recognized as compact structures of ~ 50 to 500 &#956;m in size (Figure&#160;3). We obtain ~200 islets in WT mice and 300- &#60;100 islets in db/db mice.</li>
		</ol>
		</li>
		<li>Islet Culturing
		<ol>
			<li>To help the islets recover from the enzyme digestion, culture groups of 40 islets in a 25 cm petri dish with 6ml RPMI culture media (10mM glucose) at 37 &#176;C, 5% /95% CO2/O2 for 2 days before live-cell 2-photon imaging. Change the culture media daily. </li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Live Cell 2-Photon Imaging
<ol>
	<li>Reagent Preparation
	<ol>
		<li>Extracellular Buffer
		<ol>
			<li>Prepare the sodium rich extracellular buffer for the 2-photon assay with 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 5 mM NaHCO3 and 10 mM HEPES.</li>
		</ol>
		</li>
		<li>Extracellular Dye
		<ol>
			<li>For the 2-photon assay, prepare stock SRB 8 mM in extracellular buffer, aliquot into small tubes and store at -20 &#176;C. Dilute the stock SRB 1:10 in fresh extracellular buffer to get a working concentration of 0.8 mM.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>photon Imaging
	<ol>
		<li>First, pre-incubate 2-day cultured islets in 3 mM glucose extracellular buffer for 30 min. 
		NOTE: We use islets between 2 and 5 days in culture.</li>
		<li>Then, place a glass slide onto the thermo-control chamber mounted on the microscope stage. Use grease to prevent the leaking of solution.</li>
		<li>Cover the chamber with 500 &#956;l of extracellular buffer containing 0.8 mM SRB with different glucose concentrations (3 mM, 6 mM, 15 mM and 20 mM glucose).</li>
		<li>Next, place the pre-incubated islet into the middle of the chamber and focus at 3-4 cell layers into the islet where, in rodent islets, immunostaining for insulin shows that most cells are beta-cells. 
		NOTE: The SRB will outline all the cell membrane of the islet quickly and the islet is ready to image.</li>
		<li>Use a 2-photon microscope with a 60X oil immersion objective (NA 1.42) and imaging software (e.g., ScanImage 11). Figure&#160;4 illustrates a typical microscopy arrangement. Image exocytic events using SRB as a membrane impermeant fluorescent extracellular marker excited by femtosecond laser pulses at 880 nm, with fluorescence detected at 550-650 nm. Record the islet responses to stimulation.</li>
		<li>Identify single exocytic events by the sudden appearance of a small fluorescent spot (resolution 10 pixels/&#181;m) and confirm by the fast rising phase of the fluorescent signal over a region of interest (~0.78 &#956;m2).</li>
	</ol>
	</li>
</ol>

<ol><li>Guariguata, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035%5BTitle%5D)+AND+%22Diabetes+Research+and+Clinical+Practice%22%5BJournal%5D)">Global estimates of diabetes prevalence for 2013 and projections for 2035.</a> Diabetes Research and Clinical Practice. 103, 137-149 (2014).</li>
<li>Ashcroft, F. M., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetes+mellitus+and+the+beta+cell%3A+The+last+ten+years%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Diabetes mellitus and the beta cell: The last ten years.</a> Cell. 148, 1160-1171 (2012).</li>
<li>Weir, G. C., Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Five+stages+of+evolving+beta-cell+dysfunction+during+progression+to+diabetes%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Five stages of evolving beta-cell dysfunction during progression to diabetes.</a> Diabetes. 53, S16-S21 (2004).</li>
<li>Kjorholt, C., Akerfeldt, M. C., Biden, T. J., Laybutt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+hyperglycemia%2C+independent+of+plasma+lipid+levels%2C+is+sufficient+for+the+loss+of+beta-cell+differentiation+and+secretory+function+in+the+db%2Fdb+mouse+model+of+diabetes%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Chronic hyperglycemia, independent of plasma lipid levels, is sufficient for the loss of beta-cell differentiation and secretory function in the db/db mouse model of diabetes.</a> Diabetes. 54, 2755-2763 (2005).</li>
<li>Wang, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+type+2+diabetic+rodent+models%5BTitle%5D)+AND+%22Journal+of+Diabetes+Research%22%5BJournal%5D)">Spontaneous type 2 diabetic rodent models.</a> Journal of Diabetes Research. , (2013).</li>
<li>Hummel, K. P., Dickie, M. M., Coleman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetes+a+new+mutation+in+mouse%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Diabetes a new mutation in mouse.</a> Science. 153, 1127-1128 (1966).</li>
<li>Do, O. H., Low, J. T., Gaisano, H. Y., Thorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+secretory+deficit+in+islets+from+db%2Fdb+mice+is+mainly+due+to+a+loss+of+responding+beta+cells%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells.</a> Diabetologia. 57, 1400-1409 (2014).</li>
<li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose+principally+regulates+insulin+secretion+in+mouse+islets+by+controlling+the+numbers+of+granule+fusion+events+per+cell%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell.</a> Diabetologia. 56, 2629-2637 (2013).</li>
<li>Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet.</a> Science. 297, 1349-1352 (2002).</li>
<li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+secretion+from+beta+cells+in+intact+mouse+islets+is+targeted+towards+the+vasculature%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature.</a> Diabetologia. 57, 1655-1663 (2014).</li>
<li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ScanImage%3A+flexible+software+for+operating+laser+scanning+microscopes%5BTitle%5D)+AND+%22Biomedical+Engineering+Online%22%5BJournal%5D)">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomedical Engineering Online. 2, 13(2003).</li>
</ol>

The authors declare they have no competing financial interests.

The most critical factor in the islet isolation is the initial perfusion of the pancreas; an under perfused pancreas results in a considerably lower islet yield. Other factors also affect the isolation quality such as the digestion time and the level of shaking which could partly compensate for the level of perfusion. For example, a fully perfused pancreas will need to be incubated at 37 &#176;C for ~18 min 30 sec while shaking gently, in contrast an under digested pancreas may require ~20 min&#160;digestion with harder ...

The role of beta-cell dysfunction in disease has been widely recognized 3,4. Cell lines such as the MIN6 and INS-1 are useful tools to understand the biology of beta cell behavior. However, the physiological control of insulin secretion takes place within the islets of Langerhans. These islets contain thousands of tightly packed beta cells, as well as blood vessels and other endocrine cell types. This environment within the islet influences insulin secretion and is likely to be important in diabetes. Therefore, to understand the physiological control of insulin secretion, and the pathophysiology of disease, it is essential to study intact islets.
Islet isolation
Live human islets and, in particular, human islets from type 2 diabetes patients are difficult to obtain. In addition, human islets have limited possibilities for experimental molecular manipulation. Researchers have therefore employed islets from animals and animal models of type 2 diabetes. One such disease model is the db/db mouse. This is a spontaneous mutation that models type 2 diabetes with a phenotype progression that closely parallels human disease 5,6. The protocol presented here for islet isolation from diabetic db/db mice has many steps in common with other groups with some refinements for better yield, purification and enhanced islet survival.
2 photon imaging
The live-cell 2-photon assay described here enables researchers to quantify the number and evaluate the characteristics of single insulin-containing granules from many cells of diabetic7 and wild type islets 8,9.

<table><tbody><tr><td>Liberase TL 5 mg</td><td>Roche</td><td>-5401020001</td><td></td></tr><tr><td>Collagenase type IV-1g</td><td>Gibco-Life Technologies</td><td>17104-019</td><td></td></tr><tr><td>Histopaque 1077 500 ml</td><td>Sigma Aldrich</td><td>10771</td><td></td></tr><tr><td>RPMI 1640 Medium (10x) 1L</td><td>Sigma Aldrich</td><td>R1383</td><td>to prepare isolation media</td></tr><tr><td>RPMI 1640 (1x) 500 ml</td><td>Gibco-Life Technologies</td><td>21870-076</td><td>to prepare cultured media</td></tr><tr><td>Penicillin-Streptomycin 100 ml</td><td>Gibco-Life Technologies</td><td>15140-122</td><td>to prepare cultured media&amp;nbsp;</td></tr><tr><td>Fetal bovine serum 500 ml</td><td>Gibco-Life Technologies</td><td>10099-141</td><td>to prepare cultured media</td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Gibco-Life Technologies</td><td>11966-025</td><td>to dilute the liberase</td></tr><tr><td>Metamorph program</td><td>Molecular Devices, USA</td><td></td><td>to analyze the 2-photon images</td></tr></tbody></table>

leprdb mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact 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.

Islet yield and purification
For a normal wild type mouse, about 200 islets are expected. Healthy islets look bright, round shaped and have a smooth border. An over-digested isolation batch usually has small and fuzzy islets while an under-digested batch has fewer islets and acinar cell attached islets (Figure 3). For diabetic db/db mice, the islet yield and appearance depends on the disease progression with better glycemic mice have bigger, brighter and more islets (up to 300...

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, ...

The overall goal of this procedure is to obtain viable living eyelets from the diabetic LEP R DB mice, and assess insulin secretion from beta cells within the eyelets using live cell to photon imaging. This is accomplished by first preparing reagents such as the enzyme mixture for the pancreas digestion, the culture media for eyelet culturing, and the extracellular buffer for the two photon assay. Next, the abdomen of a euthanized mouse is opened up and the enzyme mixture is injected into the pancreas to digest the organ.Then the eyelets are separated from the pancreatic digest and cultured. Finally, two photon microscopy is used to image single insulin granule fusions from beta cells within the eyelet. Ultimately, this protocol will help researchers to understand how insulin secretion is controlled in health and disease.Main refinement of the eyelet isolation technique is the use of mixed enzymes to improve the yield and the viability of eyelets. The two photon imaging technique is currently the only method to record insulin secretion from multiple cells within an intact eyelet. To prepare enzymes to isolate pancreatic eyelets dilute one vial of five milligrams of liase TL with 26 milliliters of DM EM, prepare the collagenase type four in Hank's buffer to a concentration of 0.5 milligrams per milliliter, supplemented with the following mixed liase TL and collagenase solutions.At a ratio four to one by volume aliquot the mixture of enzymes to 2.5 milliliters each and store at negative 20 degrees Celsius. To prepare RPMI isolation medium in a large conical flask containing one liter of water, dissolve one vial of RPMI 1 6 4 0 powder at room temperature and supplement with two grams of sodium bicarbonate and 4.02 grams of hippies. Use sodium hydroxide to adjust the pH to 7.4, then filter, sterilize, and store at four degrees Celsius.Make RPMI culture medium by adding 10%FBS and 1%antibiotics to the RPMI isolation medium for two photon imaging. Prepare sodium rich extracellular buffer with the following reagents. To prepare extracellular dye, make eight millimolar stock of SRB an extracellular buffer aliquot into small tubes and store at negative 20 degrees Celsius.To arrive at a working concentration, use fresh extracellular buffer to dilute the stock SRB one to 10. After sacrificing a mouse and using 70%ethanol to thoroughly spray the mouse body according to the text protocol, position the animal with the head toward and the tail away from the surgeon. Use two long lateral cuts through the abdominal skin and peritoneum to make a V-shaped flap.Pull the flap of the skin up to uncover the entire abdominal cavity. Put aside the bowel and with the folded tissue, keep the liver in place to reveal the common bile duct. Next, apply a vascular clamp at the ambula on the duodenum wall to block the injected enzymes from entering the duodenum.This is an important step that determines whether the pancreatic enzymes enter the pancreatic duct reflux, the injection site, or enter the duodenum. Use a 31 gauge needle to cannulate the common bile duct near the junction of the common hepatic duct and cystic duct and leave room for a second injection. If the pancreas fails to perfuse the first time, then slowly inject about two milliliters of cold enzyme mixture into the mouse common bile duct.If the clamp is in the right position, the pancreas will be quickly perfused. Once the pancreas is perfused, quickly remove the organ, put it into a glass vial and incubate into 37 degrees Celsius water bath for approximately 19 minutes. After the incubation, stop the digestion process by adding approximately 20 milliliters of cold isolation medium.Shake the vial gently to disrupt the pancreas and pour it through a normal T sive with a one millimeter pore size into a sterile 50 milliliter tube. Use the isolation medium to top off the tube and centrifuge at 400 G and four degrees Celsius for one minute. With the break off, add isolation medium to the palate and dissolve it.Then transfer the suspension to two 15 milliliter tubes and spin a second time. After aspirating the supinate, add six milliliters of density gradient cell separation medium to each palette and pipette or vortex to resus. Then to the side of the tubes, gently add approximately six milliliters of isolation medium on top of the density gradient separation medium centrifuge at 100 G and four degrees Celsius for 15 minutes.With the break off, collect the upper, middle and lower layers in three separate dishes. Then under a dissecting microscope, use a pipette to handpick the eyelets in the three dishes. The majority will be found in the middle layer to help the eyelets recover from the enzyme digestion.Prepare groups of 40 eyelets in 25 centimeter Petri dishes with six milliliters of RPMI culture medium containing 10 millimolar glucose and culture for two days. Changing the medium daily to carry out two photon imaging as an indirect assay to measure insulin secretion. Begin by using three millimolar glucose in extracellular buffer to pre incubate two day cultured eyelets.For 30 minutes, apply grease to the microscope stage to prevent the solution from leaking and mount thermo control chamber containing a glass slide on the microscope stage. Then with 500 microliters of extracellular buffer containing 0.8 millimolar, SRB and various glucose concentrations cover the chamber. Next, place the pre incubated eyelet into the middle of the chamber and focus at three to four cell layers into the eyelet.Where in rodent eyelets, immuno staining for insulin shows that most cells are beta cells. Then with the two photon microscope and a 60 x oil immersion objective, identify single exo acidic events by the sudden appearance of a small fluorescent spot. Finally, in the scan image software confirm an event by the fast rising phase of the fluorescent signal over a region of interest for a wild type mouse.About 200 eyelets are expected using the protocol demonstrated in this video. Healthy eyelets look bright, round shaped, and have a smooth border. And under digested isolation batch usually has small and fuzzy eyelets and asar cell attached eyelets.While an over digested batch has fewer eyelets in the two photon assay in response to stimuli such as glucose or high potassium insulin granules fsed with the plasma membrane, an SRB enters the granules, which results in the appearance of a sudden fluorescent spot, approximately 400 nanometers in diameter. Within the time recorded all the exo events in response to the stimulus are identified and the location of the events, the time of the appearance, the duration and type of the fusion. Events like KISS and Run or full fusion are characterized and are valuable for assessing any defect in the granule fusion in models of type two diabetes.This method was used to show that the defect in diabetic DB DB eyelets is the loss of full granule fusion and not a change in the kinetics of individual granule fusion. Furthermore, the biggest factor influencing decreased insulin secretion in disease is a reduction in the number of responsive cells. After watching this video, you should have a good understanding of the pancreatic perfusion and eye less actuation from the left, RDB mice, and also be able to perform the two for tongue essays to assess the incidences secretory responses within Intact isles.

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Learn pancreatic islet isolation from Lepr db mice and live-cell two-photon imaging to assess insulin secretion dynamics in type 2 diabetes research. This protocol enables real-time visualization of beta cell granule fusion events.

Lepr^db Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

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