We are extremely grateful to Ms. Jennifer Munguia for her artistic illustration of the schematic diagram. We thank Mr. Michael R. Honig at Houston&rsquo;s Community Public Radio Station KPFT for his editorial assistance. This study was supported by American Diabetes Association #1-15-BS-177 (YS), and NIH R56DK118334/R01DK118334 (YS). This work was also supported by the USDA National Institute of Food and Agriculture, Hatch project 1010840 (YS) and R01 DK095118 (SG).

All methods described here have been approved by the Animal Care and Use Committee (ACUC) of Texas A&#38;M University. The surgical tools need is shown in Figure 1 and the schematic diagram of the procedure is shown in Figure 2.
1. Solutions
<ol>
	<li>Prepare Hank&#8217;s Balanced Salt Solution (HBSS) by adding 100 mL of 10X HBSS (from stock) to 900 mL of distilled water to make 1 L HBSS (1X).</li>
	<li>Prepare STOP solution (must be made fresh and should be used within 1 h) by adding 50 mL of 100X fetal bovine serum (FBS) to 450 mL of ice cold 1X HBSS; this makes 500 mL STOP solution. Keep the STOP solution at 4 &#176;C.</li>
	<li>Prepare collagenase P solution (must be made fresh within 1 h of being used) by adding 1 mg/mL of collagenase P to ice cold 1X HBSS; use 6 mL/mouse.
	<ol>
		<li>Add 0.05% (w/v) bovine serum albumin (BSA) to collagenase P solution (this provides nutrients to the isolated islets). For example, use 3 mg BSA in 6 mL collagenase P solution. Keep on ice.</li>
		<li>Calculate and prepare the amount of collagenase P needed for all mice in one 50 mL tube.</li>
	</ol>
	</li>
	<li>Prepare complete RPMI 1640 medium by adding 10% FBS, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, INS-1 cell supplement (10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol) to 500 mL RPMI 1640 media containing 5.5&#160;mM glucose to be used for overnight culture and incubation.</li>
</ol>
2. Preparation
<ol>
	<li>Fill three 50 mL tubes of 25 mL RNase inhibiting solution, 70% ethanol or distilled H2O. These solutions will be used for cleaning surgical tools prior and during the procedure.</li>
	<li>Soak the tips of the tools in RNase inhibiting solution for 30 min before starting the protocol; this eliminates any potential RNase on the tools.</li>
	<li>Pre-cut absorbent pads to 6 in x 6 in size for use during surgery.</li>
	<li>Prepare 1X HBSS solution in advance and store at 4 &#176;C. Prepare STOP solution prior to surgery. Store at 4 &#176;C.</li>
	<li>Prepare collagenase P solution immediately prior to surgery and store on ice. 
	NOTE: This should be used within 2 h of preparation.</li>
	<li>Label 50 mL tubes for digestion and purification; prepare 2 tubes for each mouse, one for digestion and the other for islet purification. Ensure that the animal ID is on both tubes.</li>
	<li>Add 3 mL of collagenase P solution into the first 50 mL tube. The remaining 3 mL of collagenase P will be injected.</li>
	<li>Draw the remaining 3 mL of collagenase solution into a 3 mL syringe mounted with 30 G &#189; in needle. Place the syringe on ice.</li>
</ol>
3. Procedure
<ol>
	<li>Remove all the tools from RNase inhibiting solution, then dip them first in the tube with 70% ethanol, then in the tube containing distilled H2O, then air-dry on clean paper towel.</li>
	<li>Place the mouse in a chamber containing 0.5 mL of isoflurane until mouse is deeply anaesthetized.</li>
	<li>Remove mouse from the chamber and check the state of anesthesia by pinching a foot pad with forceps. Deep anesthetization is based on the observation that breathing become steadily slow and mouse is unreactive to foot pinching. After confirming that the mouse is deeply anaesthetized, euthanize the mouse with cervical dislocation, and then place the mouse on the absorbent pad.
	<ol>
		<li>Place anaesthetized mouse on its stomach, applying pressure to the neck and dislocating the spinal column from the brain by pulling tail.</li>
	</ol>
	</li>
	<li>Tape the limbs of the mouse in supine position to the absorbent pad, spray the body with 70% ethanol, and wipe excess off the excess ethanol.</li>
	<li>Use cover glass forceps and curved surgical scissors to make incisions. First make a horizontal incision on the skin of the abdominal area (~3 cm), pull the skin wide open to expose the abdominal wall. Then make a vertical incision (~3&#8211;4cm) on the abdominal peritoneum to fully expose the pancreas in abdominal cavity (Figure 3).</li>
	<li>Push the lobes of the liver superiorly to expose the bile duct, it will appear as a pale pink tube (Figure 3).</li>
	<li>Carefully move the intestines from the right lumbar/iliac region of the abdominal cavity to the right, exposing the bile duct and hepatic artery (Figure 3).</li>
	<li>Carefully clamp the common bile duct using the Schwartz micro serrefines (Figure 1) as close to the liver as possible.</li>
	<li>Identify the ampulla of Vater, which is located at the duodenal papilla, formed by the union of the pancreatic duct and the common bile duct. The ampulla of Vater appears swollen when viewed under a dissection microscope which is the entry point to the common bile duct (Figure 4). 
	NOTE: Adjusting intensity/angle of light of the dissection microscope can make it easier to locate.</li>
	<li>Insert the syringe with 3 mL of the collagenase P solution into the ampulla of Vater. Push the needle into the duct for about 1/4 of the length of the common bile duct (ampulla leads into duct) as shown in Figure 5.</li>
	<li>Once the needle is in the ampulla, ensure that the orientation of the needle is such that it is parallel with the duct.</li>
	<li>Stabilize the needle by clamping with micro Adson forceps to prevent it from puncturing the duct (Figure 5).</li>
	<li>Slowly and steadily inject 3 mL of the collagenase P solution from the syringe into the common bile duct (enough to feel resistance) as shown in Figure 6. The goal is to create backflow pressure to force the collagenase to enter the pancreatic duct. Injection is considered successful if the head, neck, body and tail region of the pancreas are all fully inflated. 
	NOTE: Islet yield will be low if either the pancreas is not fully inflated, or the splenic area is not fully inflated. The splenic area contains the highest number of islets6. Inflation can be confirmed by the appearance of open spaces between pancreatic tissue that are filled with solution.</li>
	<li>Carefully dissect out the inflated pancreas and place it in a 50 mL digestion tube containing 3 mL of ice-cold collagenase P solution.
	<ol>
		<li>Remove the pancreas using 2 forceps (curved and Micro Adson; See Figure 1): (starting from the spleen, pull the pancreas away from spleen and continue removing from the stomach and along the duodenum. 
		NOTE: No incisions required.</li>
		<li>Chop pancreas for 3&#8211;5 s in the digestion tube with 3 mL of ice-cold collagenase P solution using fine surgical scissors (Figure 7A).</li>
	</ol>
	</li>
	<li>Secure the tube to a rack in 37 &#176;C water bath and shake at 100&#8211;120 rpm for approximately 12&#8211;13 min.</li>
	<li>After incubation, gently shake the tubes by hand to disrupt the tissue until the collagenase P digestion solution becomes homogenous (Figure 7B). Homogeneity is confirmed by a sand-like appearance of fine particles of pancreas. About 30 s of gentle hand-shaking is usually enough. Hold the tube up to light to examine if the tissue is well homogenized; shake for another ~15 s if needed.</li>
	<li>Once digested, immediately place the tubes on ice and add 40 mL of ice-cold STOP solution to terminate the enzymatic digestion. 
	NOTE: At this point digestion tubes can be left on ice up to 2 h, if working on multiple mice.</li>
	<li>Centrifuge the tube in a swinging-bucket centrifuge at 300 x g for 30 s (temperature of centrifuge is flexible). 
	NOTE: Use a swinging-bucket centrifuge so that the tissue pellet is formed at the bottom of the 50 mL tube and not on the wall of the tube; this is critical for better islet yield.</li>
	<li>Decant and repeat the centrifugation with STOP solution 2 more times, decanting the solution after each spin. Use 20 mL of STOP solution for each subsequent wash.
	<ol>
		<li>Before each spin, disrupt the pellet by gently shaking the tissue pellet in the 50 mL tube in 20 mL STOP solution.</li>
	</ol>
	</li>
	<li>Re-suspend the tissue pellet with 40 mL of ice cold HBSS and centrifuge at 300 x g for 30 s.</li>
	<li>Decant and repeat the centrifugation using a swinging-bucket centrifuge with HBSS solution two more times, decanting the solution after each spin. Use 20 mL of HBSS for each wash.
	<ol>
		<li>Before each spin, disrupt the pellet, to detach it from the bottom of the tube by gently shaking the tube containing 20 mL HBSS solution.</li>
	</ol>
	</li>
	<li>After the last centrifugation remove all HBSS.</li>
	<li>Then add 5 mL of room temperature density gradient to the 50 mL tube containing the pellet. Vortex briefly at low speed until homogenized.</li>
	<li>Add another 5 mL of the room temperature density gradient to the 50 mL tube. Do not vortex/mix. 
	NOTE: It is critical to remain steady and still so as to allow better gradient to form without disruption.</li>
	<li>Pipette 10 mL of room temperature HBSS buffer into the tube (containing the density gradient), drop-by-drop gently and slowly to allow a gradient to form. Use a pipette gun with a 10 mL pipette to add HBSS dropwise.</li>
	<li>Using a swinging-bucket centrifuge, spin tubes at 1700 x g for 15 min. Make sure to change the speed of both acceleration/deceleration to the lowest setting to maintain the gradient (Figure 7C).</li>
	<li>After the spin, carefully remove the tubes without disturbing the gradient. Using a pipette gun pre-wetted with cold HBSS (pipette HBSS up and down), pipette out the layer of islets (5&#8211;10 mL) formed between the density gradient and HBSS into the new 50 mL islet collection tube. 
	NOTE: It is helpful to wet the pipette with cold HBSS prior to pipetting the islets to prevent the islets from sticking to the inner walls of the pipette.
	<ol>
		<li>In case the separation is incomplete, islets would be visible in the density gradient layer, pipette out the entire 10 mL of the density gradient (bottom layer) along with the islet layer formed at the interface (only to leave about 8&#8211;10 mL of the HBSS top layer behind). 
		NOTE: Collecting both the islet layer and the density gradient layer (bottom layer) can result in the collection of debris which may lengthen the islet picking time, but no other steps will be altered. Collecting both these layers is not necessary when islet layer is well formed.</li>
	</ol>
	</li>
	<li>Add 20 mL of ice cold HBSS to the new 50 mL tube containing islets, then centrifuge at 350 x g for 3 min in a swinging-bucket centrifuge.</li>
	<li>After the spin, carefully pipette (do not decant) out the supernatant (leave ~3 mL at the bottom) without disturbing the pellet containing the islets at the bottom. Discard the supernatant.</li>
	<li>Repeat washing and centrifuging at least 3 times. Add 20 mL of HBSS each time, and be sure to suspend the pellet before each spin.</li>
	<li>Warm the RPMI-1640 complete media bottle in a water bath at 37 &#176;C prior to use. After the last centrifugation, remove all of the HBSS and add 4 mL of previously prepared complete RPMI-1640 media (containing FBS, INS-1 cell supplement and penicillin/streptomycin) to the pellet.</li>
	<li>Dislodge the pellet by gently swirling the tube and immediately pour the RPMI-1640 media into a 100 mm Petri dish. Add another 5 mL of RPMI-1640 media to the tube and gently swirl it to wash off any remaining islets, then also pour the media into the same Petri dish.
	<ol>
		<li>Under a dissection microscope, pick healthy islets from the Petri dish using a 20 &#181;L pipette, and put them in a new Petri dish containing 10 mL of complete RPMI 1640 media. When viewed under a dissection microscope, islets should appear spherical/oblong and golden-brown color with a smooth surface compared to the relatively transparent, wispy exocrine tissue. 
		NOTE: Magnification of dissection microscope is usually set at 12.5&#8211;16x. Islet yield will vary depending on various factors including strain, age and sex of mouse. This protocol usually yields 250&#8211;350 healthy islets from healthy C57BL/6J mouse age 4&#8211;10 months old.</li>
	</ol>
	</li>
	<li>Incubate the islets in a sterile incubator at 37 &#176;C with 5% CO2 infusion and 95% humidified air overnight for experiments the next day, or freeze the islets for desired analysis at a later time.
	<ol>
		<li>Once islets are frozen, they cannot be used for secretion assays, only RNA or protein quantification. To collect cells to freeze, place them in HBSS buffer, collect all islets in 200&#8211;500 &#181;L of buffer, transfer to 1.5 mL tube, centrifuge 350 x g for 1&#8211;2 min, remove the supernatant leaving no more than 30&#8211;40 &#181;L solution, place in -20 &#176;C for short term storage (several days) or -80 &#176;C for long term storage.</li>
	</ol>
	</li>
</ol>

<ol><li>Carter, J. D., Dula, S. B., Corbin, K. L., Wu, R., Nunemaker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+practical+guide+to+rodent+islet+isolation+and+assessment%5BTitle%5D)+AND+%22Biological+Procedures+Online%22%5BJournal%5D)">A practical guide to rodent islet isolation and assessment.</a> Biological Procedures Online. 11, 3-31 (2009).</li>
<li>Gotoh, M., Maki, T., Kiyoizumi, T., Satomi, S., Monaco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+method+for+isolation+of+mouse+pancreatic+islets%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">An improved method for isolation of mouse pancreatic islets.</a> Transplantation. 40 (4), 437-438 (1985).</li>
<li>O'Dowd, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolation+and+purification+of+rodent+pancreatic+islets+of+Langerhans%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">The isolation and purification of rodent pancreatic islets of Langerhans.</a> Methods in Molecular Biology. 560, 37-42 (2009).</li>
<li>Do, O. H., Low, J. T., Thorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lepr%28db%29+mouse+model+of+type+2+diabetes%3A+pancreatic+islet+isolation+and+live-cell+2-photon+imaging+of+intact+islets%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments+%3A+JoVE%22%5BJournal%5D)">Lepr(db) mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets.</a> Journal of Visualized Experiments : JoVE. (99), e52632(2015).</li>
<li>Stull, N. D., Breite, A., McCarthy, R., Tersey, S. A., Mirmira, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+islet+of+Langerhans+isolation+using+a+combination+of+purified+collagenase+and+neutral+protease%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments+%3A+JoVE%22%5BJournal%5D)">Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease.</a> Journal of Visualized Experiments : JoVE. (67), (2012).</li>
<li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regional+Differences+in+Islet+Distribution+in+the+Human+Pancreas+-+Preferential+Beta-Cell+Loss+in+the+Head+Region+in+Patients+with+Type+2+Diabetes%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">Regional Differences in Islet Distribution in the Human Pancreas - Preferential Beta-Cell Loss in the Head Region in Patients with Type 2 Diabetes.</a> PLOS ONE. 8, (2013).</li>
<li>Li, D. S., Yuan, Y. H., Tu, H. J., Liang, Q. L., Dai, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+protocol+for+islet+isolation+from+mouse+pancreas%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">A protocol for islet isolation from mouse pancreas.</a> Nature Protocols. 4 (11), 1649-1652 (2009).</li>
<li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments+%3A+JoVE%22%5BJournal%5D)">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of Visualized Experiments : JoVE. (88), e50374(2014).</li>
<li>Scharp, D. W., Lacy, P. E., Finke, E., Olack, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-temperature+culture+of+human+islets+isolated+by+the+distention+method+and+purified+with+Ficoll+or+Percoll+gradients%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">Low-temperature culture of human islets isolated by the distention method and purified with Ficoll or Percoll gradients.</a> Surgery. 107 (5), e50374(1987).</li>
<li>Salvalaggio, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Islet+filtration%3A+a+simple+and+rapid+new+purification+procedure+that+avoids+ficoll+and+improves+islet+mass+and+function%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Islet filtration: a simple and rapid new purification procedure that avoids ficoll and improves islet mass and function.</a> Transplantation. 74 (66), 877-879 (2002).</li>
<li>Saliba, Y., Bakhos, J. J., Itani, T., Fares, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+optimized+protocol+for+purification+of+functional+islets+of+Langerhans%5BTitle%5D)+AND+%22Laboratory+Investigation%22%5BJournal%5D)">An optimized protocol for purification of functional islets of Langerhans.</a> Laboratory Investigation. 97 (1), 70-83 (2017).</li>
<li>Pradhan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Obestatin+stimulates+glucose-induced+insulin+secretion+through+ghrelin+receptor+GHS-R%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Obestatin stimulates glucose-induced insulin secretion through ghrelin receptor GHS-R.</a> Scientific Reports. 7 (1), 979(2017).</li>
<li>Shapiro, A. M. J., Hao, E., Rajotte, R. V., Kneteman, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+yield+of+rodent+islets+with+intraductal+collagenase+and+stationary+digestion--a+comparison+with+standard+technique%5BTitle%5D)+AND+%22Cell+Transplantation%22%5BJournal%5D)">High yield of rodent islets with intraductal collagenase and stationary digestion--a comparison with standard technique.</a> Cell Transplantation. 5 (6), 631-638 (1996).</li>
</ol>

The authors have nothing to disclose.

This protocol includes collagenase perfusion and digestion, followed by purification of islets. The most critical steps of this protocol are effective injection and complete perfusion of the pancreas1,4,7. The delivery method of this protocol allows the enzyme to traverse the anatomical routes to better digest the exocrine tissue surrounding the islets1. In addition, this technique is well suited for comp...

There are two common methods in the literature for pancreatic islet isolation. One requires excising the pancreas and dicing it into small pieces using surgical scissors, and then digesting it in a collagenase solution1,2,3. Another more precise method is to use the network of ducts present in the pancreas to introduce digestive enzyme. The following sites have been used for digestive enzyme injection: the junction of the bile and cystic duct, the gallbladder into the common bile duct, or the common bile duct itself1,4,5. It is known that islets are not evenly distributed in the pancreas; the splenic region contains the most islets6. While the second method using anatomical routes to deliver digestive enzymes allows for a more complete perfusion of the pancreas, including the splenic region, this procedure often requires clamping or suturing of the ampulla of Vater that is technically challenging. In terms of islet purification, multiple density gradients, as well as cell strainers and magnetic retraction have been used to purify the islets3,7. The utilization of these gradients can be time consuming and the Ficoll gradients can result in toxic damage of islets8.
The current protocol is built on the method described by Li et al.7, with additional modifications added based on the experience of ourselves and others1,4. The most critical steps of our protocol are clamping of the common bile duct near the liver end, injecting collagenase P via the ampulla of Vater to digest the exocrine tissue, and then using a shaking water bath to expedite the digestion mechanically1,4,7. Subsequently, a 'STOP' solution is applied to inhibit further digestion of the islets; HBSS is used to wash off the remaining collagenase P and STOP solution. When the Ficoll method was used to purify human islets, yield was reported to be twice the islets with greater functional capability (e.g., insulin secretion) as compared to the use of Percoll gradients9. However, studies have questioned the use of Ficoll gradient due to its toxic effect on the islets1,10. It has been reported that the Histopaque gradient provides optimal purification kinetics for mouse islet isolation, which produces good yield of high quality islets with simpler steps and lower cost1. In our protocol, Histopaque-1077 is used to purify islets from other residual tissue8,11. The harvested islets can be cultured in complete RPMI-1640 media, or directly utilized in RNA/protein quantitation.
Our protocol, using a combination of collagenase P digestion and a single gradient purification step, is simpler than other published protocols. Our method does not require demanding surgical procedures and has just a few simple steps. More importantly, this protocol consistently produces a good yield of high quality functional islets (250-350/mouse) as we reported12.

<table><tbody><tr><td>3 mL syringe</td><td>BD</td><td>309657</td><td>Hoding collagenase P</td></tr><tr><td>Coverglass forceps</td><td>VWR</td><td>82027-396</td><td>Holding skin of mouse to aid incision procedure</td></tr><tr><td>Curved forceps</td><td>Sigma-Aldrich</td><td>Z168696</td><td>Holding tissues during pancreas removal</td></tr><tr><td>Isoflurane</td><td>Piramal</td><td>B13B16A</td><td>To anaesthetize mice prior surgery</td></tr><tr><td>100 mm petri dishes</td><td>VWR</td><td>30-2041</td><td>Used for islet culture</td></tr><tr><td>30 G. &amp;frac12; inch needle</td><td>BD</td><td>305106</td><td>For penetration of Ampulla of vater to deliver Collagenase P - this guage is used as it fits well in most CBDs</td></tr><tr><td>50ml tube</td><td>VWR</td><td>89039-658</td><td>Holding digested pancreatic tissue, collagenase P, and purified islets</td></tr><tr><td>Absorbent pads with waterproof moisture barrier</td><td>VWR</td><td>82020-845</td><td>To absorb blood from syurgical procesdudes</td></tr><tr><td>Centrifuge 5810R with swing bucket and deceleration capability</td><td>Eppendorf</td><td>5811FJ478114</td><td>Use for pelleting tissues, pellet is formed at bottom of conical tube - swing bucket centrifuge is needed. Also the decelaration feature is important to form the gradient layers.</td></tr><tr><td>Collagenase P- 1g</td><td>Roche Diagnostics</td><td>11249002001</td><td>For digestion of exocrine pancreas</td></tr><tr><td>Curved surgical scissors</td><td>Fisher-Scientific</td><td>13-804-21</td><td>For cutting open mouse abdomen</td></tr><tr><td>Dissection microscope</td><td>Olympus</td><td>SZX16</td><td>Used for identification of key anatomical structures to accurately deliver collagenase into pancreas</td></tr><tr><td>Hank&#039;s Balanced Salt Solution 10x</td><td>Corning</td><td>20-023-CV</td><td>Washing cells</td></tr><tr><td>Histopaque-1077</td><td>Sigma</td><td>RNBF5100</td><td>For gradient formation</td></tr><tr><td>Light source</td><td>Leeds</td><td>LR92240</td><td>Enhancing visibility of microscope</td></tr><tr><td>RNaseZap</td><td>Fisher-Scientific</td><td>AM9780</td><td>For removing RNase</td></tr><tr><td>RPMI-1640 Media w/o L-Glutamine</td><td>Corning</td><td>15-040-CV</td><td>Culturing Islets</td></tr><tr><td>Schwartz micro serrefines (Microvascular clamp)</td><td>Fine Science Tools</td><td>18052-01</td><td>Clamping common bile duct and hepatic artery</td></tr><tr><td>Shaking waterbath</td><td>Boekel/Grant</td><td>8R0534008</td><td>Important for mechanical digestion of exocrine tissue</td></tr><tr><td>Small surgical scissors</td><td>VWR</td><td>82027-578</td><td>Cuttitng tissue that atached to pancreas</td></tr></tbody></table>

a simple high efficiency protocol for pancreatic islet isolation from mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

Proper completion of this procedure requires some understanding of mouse anatomy in the abdominal cavity. This allows for proper identification of the ampulla of Vater and clamping of the common bile duct. The entire procedure normally takes 1–2 h. It is more efficient to isolate islets from 4–6 mice at the same time, so several samples can be centrifuged together. The time for islet-picking varies, depending on the number of islets and the efficiency of digestion; it may take roughly an hour to pick 250–350 islets...

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A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

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29.2K Views.  Texas A&M University. Dit protocol vereist het gebruik van een enkele dichtheid gradiënt waardoor het aanzienlijk minder arbeidsintensief en kosteneffectiever in vergelijking met meer complexe conventionele eilandje isolatie methoden. Het belangrijkste voordeel van dit protocol is dat het enzym rechtstreeks aan de alvleesklier wordt geleverd, wat resulteert in een verbeterde weefselvertering en verhoogde eilandjesopbrengst. Begin met het aftappen van de ledematen van de muis in de supine positie en spuit het lich...

Video: A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Medicine

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Developmental Biology

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

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 characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have 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. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

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 I: digestion and collection of pancreatic tissue) 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 ...

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.

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

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

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

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

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 ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

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 ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

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