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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus+and+the+beta+cell:+The+last+ten+years.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+stages+of+evolving+beta-cell+dysfunction+during+progression+to+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+type+2+diabetic+rodent+models.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+a+new+mutation+in+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretory+deficit+in+islets+from+db/db+mice+is+mainly+due+to+a+loss+of+responding+beta+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+principally+regulates+insulin+secretion+in+mouse+islets+by+controlling+the+numbers+of+granule+fusion+events+per+cell.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion+from+beta+cells+in+intact+mouse+islets+is+targeted+towards+the+vasculature.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">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 shaking. The mixture of two digestion enzymes in our hands provides better islet isolation than either one alone. For the collagenase only method, the digestion time critically affects the islet yield and time differences of only 30 sec can dramatically affect quality. For liberase only, the yield is acceptable but under-digestion sometimes happens, we believe that the addition of collagenase helps to digest the connective tissue better.
We have found the db/db mice have diverse diabetic phenotypes, even among siblings. As a result, glucose tolerance tests on each animal before sacrificing are routinely performed. This enables classification the animals in terms of resting glucose levels and the area under the curve in response to injected glucose.
There are a number of possible experiments that can be performed after the islets have been isolated. Isolated islets can be fixed in paraformaldehyde or methanol immediately after isolation and used in immunofluorescence staining to determine the location of proteins of interest 10. Isolated islets can be dispersed in to single cells. This can be done on the same day of islet isolation or after a few days of islet culture. The single cells are then plated out and it is this preparation that has been the mainstay for techniques such as patch clamp and total internal reflection microscopy. In experiments that measure insulin secretion, as well as for live-cell 2-photon imaging, we have found that islets require 2 days in culture in order to give reproducible data. What is happening in culture is not well understood but is thought to be a period of recovery from the isolation and digestion process.
The 2-photon technique presented here enables researchers to image the insulin secretory process at the level of individual granule fusion from many cells within intact islets. We have found that the number of granule fusion events and their timing is consistent with the amounts and temporal profile of insulin secretion 8. This suggests the method is detecting the majority of insulin granule fusion events. Given that dye entry in to the fusing granules is not a specific method for detecting insulin granule fusion events we have gone to great lengths to conduct control experiments to demonstrate that insulin granule fusion in beta cells are being studied 7,8. However, a component of this evidence is dependent on the structure of the rodent islet where beta cells are enriched in the core of the islet. The different structure of human islets, where different endocrine cells are scattered throughout the islet volume, would require additional methods to identify beta cells.
There are a number of technical problems with the 2-photon microscope that are generic to all imaging approaches. For example, we carefully balance the opposing factors of trying to get enough light in to the islets so that we can reduce the gain of the detectors, and therefore reduce noise, with putting so much light in to the islets that we cause cell damage. In our system, a Pockels cell is used to attenuate the pulsed laser (Figure&#160;3); all commercial systems have mechanisms to alter the intensity of light that falls on to the specimen.
In summary, the combination of careful isolation of db/db islets and whole-islet 2 photon microscopy present new opportunities to study the processes of insulin secretion and determine the key defective elements in type 2 diabetes.

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

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&#160;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 islets) compared to worse glycemic mice which have smaller islets with a translucent appearance (under 100 islets)
2-photon assay
2-photon imaging is an indirect assay to measure insulin secretion at the level of single insulin granule fusion. In response to stimuli such as glucose or high potassium, insulin granules fuse with the plasma membrane and the extracellular dye SRB enters the granules which results in the appearance of a sudden fluorescent spot ~400 nm in diameter (Figure&#160;5). Various experiments have been carried out to validate this assay as a measurement of fusion of insulin-containing granules like the glucose dose dependent of increasing number of granule fusion events with the expected amount of insulin secretion, the consistent size of the responded cells in the 2 photon with that of a beta cell, the similar granule diameter measured by the 2 photon and the electron microscope, the colocalization of the dye uptake and insulin staining 8.
Within the time of recording, all the exocytic 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 (Figure&#160;5, 6). These characteristics of the fusion process are valuable for assessing any defect in the granule fusion in models of type 2 diabetes. Our previous report using this method shows that the defect in diabetic db/db islets is the loss of full granule fusion and not a change in the characteristics of the kinetics of individual granule fusion 7. Furthermore, we show that the biggest factor in the decreased in insulin secretion in disease is a reduction in the number of responsive cells 7.
<img alt="Figure 1" src="/files/ftp_upload/52632/52632fig1.jpg" /> 
Figure 1: A diagram of the sites of clamping and injection. Inject the enzymes to the common bile duct near the junction of the common hepatic duct and cystic duct. Clamp the ampulla of Valter by a vascular clamp to facilitate the injected enzyme to the pancreatic duct.
<img alt="Figure 2" src="/files/ftp_upload/52632/52632fig2.jpg" /> 
Figure 2: The injection process in the mice. Top figure illustrates the needle inside the common bile duct with a vascular clamp applied at the ampulla of Valter. Bottom figure shows the liberase-perfused pancreas after injection.
<img alt="Figure 3" src="/files/ftp_upload/52632/52632fig3.jpg" /> 
Figure 3: Typical images of Wild type and db/db isolated islets. (A) Wild type isolated islets. (B) db/db isolated islets. (C) Under-digested islets with attached acinar cells (arrows). (D) Over-digested islets with small and fuzzy islets (arrows). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52632/52632fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52632/52632fig4.jpg" /> 
Figure 4: The main components of the custom-made 2 photon microscope. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52632/52632fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52632/52632fig5.jpg" /> 
Figure 5: Example of a single insulin granule fusion event as recorded with 2 photon microscopy. Left figure represents the cell before (A) and after (B) the appearance of an exocytic event (arrow) in response to 15 mM glucose stimulation. (C) illustrates the SRB labeling with the entering of the extracellular dye SRB into the granule when it fuses with the plasma membrane. (D) shows the fluorescence intensity over a region of interest of an exocytic event as well as the sequential images of this exocytic event (scale bar 1 &#956;m). Image modified from Do et al 2014 7. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52632/52632fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52632/52632fig6.jpg" /> 
Figure 6: Responses of the wild type and diabetic db/db islets under the 2-photon microscope with SRB as the extracellular dye. In these experiments insulin granule fusion events in response to 15mM glucose stimulation are recorded. Yellow circles are sites of the exocytic events during 20 min of recording. Image modified from Do et al 2014 7.

Watch this Scientific Journal Video about Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets at JoVE.com

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.

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, ...

leprdb-mouse-model-type-2-diabetes-pancreatic-islet-isolation-live

Universidad San Sebastian

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Medicine

9.4K Views.  The University of Queensland. 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.

Video: Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Biology

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

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

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

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

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

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

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

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).

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 ...

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 ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Grafting Pancreatic Islets: A Method to Implant Pancreatic Islets into the Anterior Chamber of the Mouse Eye for In Vivo Imaging

Source: Nilsson, J.et. al.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/v/61234/" target="_blank">A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber</a>. J. Vis. Exp. (2020).
This video describes the protocol for grafting human pancreatic islets into the anterior chamber of the mouse&#8217;s eye to monitor revascularization of human islet grafts in vivo. It also helps to understand the contribution of recipient versus donor cells in promoting the encapsulation and vascularization of the graft.

Szczepienie wysp trzustkowych: metoda wszczepiania wysp trzustkowych do przedniej komory oka myszy w celu obrazowania in vivo

Source: Nilsson, J.et. al.&#160;A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the ...

JoVE Encyclopedia of Experiments

Cancer Research

Pancreatic Cancer

Assays & techniques

Intravital Imaging: A Method for Cellular Level Observation of the Mouse Pancreas Through a Stabilized Imaging Window

Source: Park, I. and Kim, P. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62538">Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window.</a> J. Vis. Exp. (2021).
In this video, we describe the intravital imaging of a mouse pancreas through a stabilized window for long-term visualization of cellular dynamics.

Obrazowanie przyżyciowe: metoda obserwacji trzustki myszy na poziomie komórkowym przez ustabilizowane okno obrazowania

Source: Park, I. and Kim, P. Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital ...

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

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Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

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

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

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

Szczepienie wysp trzustkowych: metoda wszczepiania wysp trzustkowych do przedniej komory oka myszy w celu obrazowania in vivo

Obrazowanie przyżyciowe: metoda obserwacji trzustki myszy na poziomie komórkowym przez ustabilizowane okno obrazowania