Name: a simple high efficiency protocol for pancreatic islet isolation from mice
Uploaded: 2019-08-30T12:30:00
Description: Watch this Scientific Journal Video about A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice at JoVE.com

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 b...

<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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+rodent+islet+isolation+and+assessment.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+isolation+of+mouse+pancreatic+islets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+purification+of+rodent+pancreatic+islets+of+Langerhans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lepr(db)+mouse+model+of+type+2+diabetes:+pancreatic+islet+isolation+and+live-cell+2-photon+imaging+of+intact+islets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+islet+of+Langerhans+isolation+using+a+combination+of+purified+collagenase+and+neutral+protease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+Differences+in+Islet+Distribution+in+the+Human+Pancreas+-+Preferential+Beta-Cell+Loss+in+the+Head+Region+in+Patients+with+Type+2+Diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+islet+isolation+from+mouse+pancreas.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-temperature+culture+of+human+islets+isolated+by+the+distention+method+and+purified+with+Ficoll+or+Percoll+gradients.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+filtration:+a+simple+and+rapid+new+purification+procedure+that+avoids+ficoll+and+improves+islet+mass+and+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+protocol+for+purification+of+functional+islets+of+Langerhans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obestatin+stimulates+glucose-induced+insulin+secretion+through+ghrelin+receptor+GHS-R.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+yield+of+rodent+islets+with+intraductal+collagenase+and+stationary+digestion--a+comparison+with+standard+technique.">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>

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. &#189; 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&#39;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&#8211;2 h. It is more efficient to isolate islets from 4&#8211;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&#821...

Watch this Scientific Journal Video about A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice at JoVE.com

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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