This work was supported by grant from NIDDK (DK065949 for GG). We thank Dr. Brenda M. Jarvis and Jeff Duryea Jr. for reading the manuscript.

Animal usage follows the procedures specified in protocol M/11/181 approved by the Vanderbilt Institutional Animal Care and Use Committee for Gu. CD1 or CBA/Bl6 mice were purchased from commercial vendors and crossed in the Vanderbilt animal facility to obtain neonatal mice.
1. Preparation of Mice, Stock Solutions, and Equipment
<ol>
	<li>For the mouse cross: set up a mouse cross and record the plugging dates to aid experimental planning. CD1 mice usually give birth around day 19 after mating. Use CD1 or CBA/Bl6 crosses to obtain CD1 or CBA/Bl6 neonates, respectively. Intercrosses between the two lines utilize CD1 females and CBA/Bl6 males.</li>
	<li>For the collagenase stock: weigh 200 mg collagenase Type IV with a high precision balance. Transfer to a 50 mL centrifuge tube. Dissolve in 40 mL HBSS with Ca2+/Mg2+ (1.26 and 0.5 mM, respectively) to make a 5 mg/mL stock solution.
	<ol>
		<li>Allow collagenase to dissolve for ~ 30 min with gentle shaking. Aliquot and keep the solution frozen in -20 &#176;C as stock, which stays active for at least one year.</li>
	</ol>
	</li>
	<li>Autoclave to sterilize several pairs of tweezers, scissors, and P20 tips for dissection and islet picking.</li>
	<li>For the 1 M glucose stock: weigh 9 g glucose in a 50 mL centrifuge tube, add 50 mL RPMI 1640 media to dissolve the glucose. Keep in 4 &#176;C until use, but no more than 6 months.</li>
</ol>
2. Working Solutions
NOTE: The day of islet isolation, prepare the following reagents.
<ol>
	<li>For the collagenase type IV working solution: thaw and dilute collagenase stock on ice. Dispense 0.15 mL stock into each 1.7 mL microfuge tube. Add 1.35 mL HBSS with Ca2+/Mg2+. Invert the tube 5 times. Leave on ice for 15 min.
	<ol>
		<li>Spin at high speed for 3 min in a microcentrifuge. Transfer supernatant to a new tube. Leave on ice until use. Discard the tube with insoluble debris.</li>
	</ol>
	</li>
	<li>For complete RPMI 1640 media: add 2.5 mL 1 M glucose, 50 mL heat inactivated FBS, and 5 mL 100x Pen-strep stock into 500 mL RPMI 1640 media. Leave on ice until use.</li>
</ol>
3. Pancreas Isolation and Digestion
<ol>
	<li>Euthanize neonates with isoflurane, followed by decapitation according to approved protocols.</li>
	<li>Lay the mouse with its belly facing up. Spray with 70% ethanol for sterilization.</li>
	<li>Lift the belly skin up with tweezers and cut the skin and muscle layers longitudinally along the midline with a pair of scissors, from the genital area to the rib cage. This opens the abdominal cavity to expose all internal organs.</li>
	<li>Transfer all the internal organs to a 100 cm dish with a pair of tweezers. Locate the pancreas, which is a scattered-organ with a dorsal portion that clings to the stomach and spleen and a ventral portion that clings to the duodenum11.</li>
	<li>Add HBSS into the dish to submerge the organs. In solution, the pancreatic tissues no longer cling to the duodenum, stomach, or spleen. It is readily recognizable under a dissecting scope because of its typical white color. Use a pair of tweezers to peel the pancreatic tissues away from surrounding tissue and transfer to a new 60 mm dish with HBSS.</li>
	<li>Cut each pancreas with a scissor into pieces smaller than 5 mm across any dimensions. Transfer to a 1.5 mL microcentrifuge tube. Up to five pancreata (younger than P7) can be digested in one tube. For P8-P16 mice, use one tube per pancreas.</li>
	<li>Add 0.5 - 1 mL collagenase working solution to each tube (0.2 mL for each P1-P7 pancreas. Use 0.5 mL for each P8-P16 pancreas). Leave the tube in a 37 &#176;C incubator for up to 15 min. Invert the tube two times every min.</li>
	<li>After ~ 5 min, tap the tube to monitor the digestion status of the pancreatic tissue. Continue the digestion process until most of the pancreatic tissue appears as fragmented cell clusters, with diameter &#60; 2 mm. The lysate will appear cloudy, which is due to normal acinar cell autolysis.</li>
</ol>
4. Lysate Washing
<ol>
	<li>Spin the lysate at 500 x g for 10 s in a microcentrifuge. Remove the supernatant layer with a P1,000 pipetman. The supernatant should appear turbid but devoid of cell clusters. Do not use aspiration, which may quickly aspirate away the tissue fragments at the bottom due to the presence of some sticky DNA.</li>
	<li>Add 1 mL RPMI 1640 complete media. Tap the tube gently to resuspend the fragmented lysate. Invert up and down 10 times. Repeat step 4.1.</li>
	<li>Repeat step 4.2 two more times or until the supernatant appears clear. Re-suspend washed pancreatic fragments in 1 mL complete RPMI 1640 media.</li>
</ol>
5. Islet Isolation
NOTE: For small numbers of pancreata, direct hand-picking as below (5.1) can be used for islet isolation. For large numbers of pancreata (&#62; 6), the method outlined in 5.2 is preferred.
<ol>
	<li>Direct hand-picking:
	<ol>
		<li>Transfer lysate from step 4.3 to a 60 mm dish. Add 5 mL complete RPMI 1640 media. Bring under a dissecting microscope.</li>
		<li>Set up the bright field illumination on the microscope. Adjust the light intensity to ~ 50% of the maximum power. Under this condition, islets appear as slightly pink clusters, whereas acinar cells as dark irregular-shaped clusters (Figure 1A). A magnification of ~ 50X (combining the power of the objective and the eye piece) is recommended to offer the best way of visualizing islets and the pipet tip opening simultaneously for picking.</li>
		<li>Pick up islets with a P20 pipette tip while avoiding acinar clusters. Dispense islets-enriched fractions to a new 60 mm dish with RPMI complete media (Figure 1B).</li>
		<li>Repeat the hand-picking process twice more. Leave pure islets in a new 60 mm dish with 4 mL complete RPMI media (Figure 1C).</li>
	</ol>
	</li>
	<li>Gradient centrifugation:
	<ol>
		<li>Prepare a 15 mL centrifuge tube that is preloaded with 2 mL polysucrose and sodium diatrizoate at a density of 1.077 g/mL. Leave the tube on ice until use.</li>
		<li>Transfer the suspension from step 4.3 onto the top of the polysucrose and sodium diatrizoate solution with a Pasteur pipette. Do not disturb the bottom layer. Tissues from up to 10 newly born or 2 P17 pancreata can be loaded into each 15 mL tube.</li>
		<li>Spin the tube at ~ 600 x g in a swing rotor for 15 min at 4 &#176;C. Use brake-off settings.</li>
		<li>After centrifugation, transfer the media and interphase layers (note that under ideal operation conditions, the islets will be located in the interphase between the polysucrose and culture media after centrifugation) into a new 15 mL tube. Add 10 mL media, mix gently but thoroughly.</li>
		<li>Spin down the islet-enriched fraction at 300 x g for 5 min. Remove the wash media. Repeat the wash one more time with complete RPMI media. Resuspend pellets in to 4 mL media, which contains mostly islets and ducts (Figure 1D).</li>
		<li>Hand-pick the islets as outlined in section 5.1.</li>
	</ol>
	</li>
</ol>
6. GSIS Assays in Isolated Islets
<ol>
	<li>Incubate the islets from step 5.1.4 in complete RPMI media for 2 h in a 37 &#176;C tissue culture incubator.</li>
	<li>Use a pipette to remove RPMI under a dissecting microscope. Add 3 mL KRB solution with 2.8 mM glucose to the plate3. Swirl 10 times to wash the islets. Remove KRB solutions.
	<ol>
		<li>Repeat the washing process once. Incubate the islets with 3 mL KRB solution at 37 &#176;C for 1 h in an incubator.</li>
	</ol>
	</li>
	<li>Aliquot 1 mL KRB into each well of a 12-well plate. Pre-warm the KRB stock and plate to 37 &#176;C in an incubator.</li>
	<li>Wash the islets twice with pre-warmed KRB as in 6.2. Transfer 10 islets to each well of the pre-warmed plates. Incubate in a 37 &#176;C incubator.</li>
	<li>After 45 min, withdraw 50 &#181;L of supernatant. This contains the insulin solution secreted under basal glucose.</li>
	<li>Add 32 &#181;L 500 mM glucose. Swirl the plate 20 times to mix the solution. Incubate in a 37 &#176;C incubator. Swirl the plate 5 times once every 5 min.</li>
	<li>After 45 min, withdraw 50 &#181;L supernatant. This is the insulin secreted under high glucose.</li>
	<li>Transfer all islets to a 1.5 mL tube. Freeze in -20 &#176;C for 30 min. Thaw at room temperature for 5 min. Repeat the freezing and thawing process.</li>
	<li>Add 0.5 mL 70% ethanol with 20 mM HCl. Leave overnight. This is the insulin left in islets.</li>
	<li>Use conventional ELISA to assay insulin levels, described previously3.</li>
</ol>

<ol>
	<li>Matsumoto, S., 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=Improvement+of+pancreatic+islet+cell+isolation+for+transplantation.">Improvement of pancreatic islet cell isolation for transplantation.</a> Proc (Bayl Univ Med Cent). 20 (4), 357-362 (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <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+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J Vis Exp. (50), (2011).</li><li>Zhao, A., 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=Galphao+represses+insulin+secretion+by+reducing+vesicular+docking+in+pancreatic+beta-cells.">Galphao represses insulin secretion by reducing vesicular docking in pancreatic beta-cells.</a> Diabetes. 59 (10), 2522-2529 (2010).</li><li>Planas, R., 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=Gene+expression+profiles+for+the+human+pancreas+and+purified+islets+in+type+1+diabetes:+new+findings+at+clinical+onset+and+in+long-standing+diabetes.">Gene expression profiles for the human pancreas and purified islets in type 1 diabetes: new findings at clinical onset and in long-standing diabetes.</a> Clin Exp Immunol. 159 (1), 23-44 (2010).</li><li>Tonne, J. M., 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+gene+expression+profiling+of+pancreatic+islets+in+mice+during+streptozotocin-induced+beta-cell+damage+and+pancreatic+Glp-1+gene+therapy.">Global gene expression profiling of pancreatic islets in mice during streptozotocin-induced beta-cell damage and pancreatic Glp-1 gene therapy.</a> Dis Model Mech. 6 (5), 1236-1245 (2013).</li><li>London, N. J., Swift, S. M., Clayton, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+functional+evaluation+of+islets+of+Langerhans.">Isolation, culture and functional evaluation of islets of Langerhans.</a> Diabetes Metab. 24 (3), 200-207 (1998).</li><li>Rorsman, P., 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=Failure+of+glucose+to+elicit+a+normal+secretory+response+in+fetal+pancreatic+beta+cells+results+from+glucose+insensitivity+of+the+ATP-regulated+K++channels.">Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels.</a> Proc Natl Acad Sci U S A. 86 (12), 4505-4509 (1989).</li><li>Hellerstrom, C., Swenne, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+and+proliferation+of+fetal+pancreatic+beta-cells.">Functional maturation and proliferation of fetal pancreatic beta-cells.</a> Diabetes. 40 (Suppl 2), 89-93 (1991).</li><li>Blum, B., 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=Functional+beta-cell+maturation+is+marked+by+an+increased+glucose+threshold+and+by+expression+of+urocortin+3.">Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3.</a> Nat Biotechnol. 30 (3), 261-264 (2012).</li><li>Hegre, O. D., 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=Nonenzymic+in+vitro+isolation+of+perinatal+islets+of+Langerhans.">Nonenzymic in vitro isolation of perinatal islets of Langerhans.</a> In Vitro. 19 (8), 611-620 (1983).</li><li>Dolen&#353;ek, J., Rupnik, M. A., Sto&#382;er, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+similarities+and+differences+between+the+human+and+the+mouse+pancreas.">Structural similarities and differences between the human and the mouse pancreas.</a> Islets. 7 (1), e102445 (2015).</li><li>White, J., White, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Source Book of Enzymes, Source Book of Enzymes. , (1997).</li><li>Bock, T., Pakkenberg, B., Buschard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+the+size+and+structure+of+the+endocrine+pancreas.">Genetic background determines the size and structure of the endocrine pancreas.</a> Diabetes. 54 (1), 133-137 (2005).</li><li>Bouwens, L., Rooman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pancreatic+beta-cell+mass.">Regulation of pancreatic beta-cell mass.</a> Physiol Rev. 85 (4), 1255-1270 (2005).</li></ol>

The authors declare no competing financial interests for the described work.

Here, we provide a step-by-step protocol on islet isolation from pancreata that are too small for conventional perfusion. It is expected to yield islets ready for all islet-based studies such as beta-cell purification, gene expression analysis, islet beta-cell maturation, proliferation, cell stress responses, cell survival, metabolism, and functional GSIS maintenance, etc. This will be, to the best of our knowledge, the first detailed visual protocol that guides new researchers to perform islet isolation from neonatal mice. Without these visual aides, extensive trial and error is expected for most researchers to achieve successful islet isolation from small pancreata.
The most critical factor for a successful islet isolation is the proper degree of pancreatic digestion as outlined in steps 3.6 - 3.8. In general, both under- and over-digestion reduce islet yield. Over-digestion further compromises islet architecture, which makes them unsuited for functional assays. To achieve best digestion results, the reaction has to be constantly monitored to visualize the tissue fragmentation process. Counting on the duration of digestion to judge the acinar-islet separation is the least dependable method, because each batch of collagenase is different and the age/size of the starting pancreas profoundly impacts the digestion process. In case of under-digestion, large endocrine-exocrine clusters can be washed in HBSS and digested again with collagenase. The digestion can then be monitored directly under a dissecting microscope to determine the time of continued digestion. Gentle pipetting can also be utilized to aide tissue dissociation. In this case, a P1,000 tip with wide opening needs be utilized, which lessens the shearing force that could destroy the islet architecture. Over-digested islets are not recommended for most subsequent studies, but can be used for some assays, such as gene expression analysis with careful controls.
Beside the above caution, paying attention to several procedural factors can further improve the final islet yield and quality. First, Ca2+ and Mg2+ should be included in all solutions, if non-commercial sources were utilized. These cations are necessary to maintain islet integrity. Second, fresh collagenase solution with high activity is essential. Collagenase solution with low activity results in a longer time for tissue dissociation. This causes substantial exocrine cell autolysis and islet destruction. To this end, utilizing collagenase stocks with more than three-rounds of freezing-thawing is not recommended. Third, low speed centrifugation during tissue washing is recommended. The rule of thumb is to use a g force (&#60; 500 x g) that is sufficient to sediment the cell clusters while maintain cell debris in the supernatant in step 4.1 of the protocol. This speed not only avoids destroying islets, but also helps to remove cell debris to enable easier islet visualization during hand picking. Lastly, collagenase cannot be inactivated by serum 12. Thus, its removal by washing is essential to ensure islet integrity in later studies.
Finally, it should be noted that the presented protocol should be limited to mouse pancreata from P1 to P17. It does not work well for pancreata older than P18. Conventional perfusion for these older mice is recommended for better yields and healthier islets. Moreover, the genetic background and age of mice profoundly affect the islet yield 13,14. Therefore, even optimal conditions will yield different number of islets per pancreas, which is normal and expected.

Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors 1-3. It is also necessary to establish endocrine gene expression in islet cells 4, 5. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (6 and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.
Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability 7, 8. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures 7, 9, 10. However, detailed description of the islet isolation process with visual aid is lacking 7, 9, making it a challenge for most researchers to perform similar studies. 
We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Collagenase&#160; Type IV</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:128px;">C5138</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:169px;">1X HBSS with Ca2+/Mg2+</td>
			<td style="width:253px;">Mediatech/Cellgro, Manassas, VA</td>
			<td>MT21020CV</td>
			<td style="width:219px;">If HBSS wihout Ca2+/Mg2+ is obtained, CaCl2 and MgSO4 can be added to 1.26 and 0.5 mM, respectively.&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:169px;">RPMI 1640 w/o glucose</td>
			<td style="width:253px;">Thermo Fisher Scientific/Life Technologies, Waltham, MA</td>
			<td>11879-020</td>
			<td style="width:219px;">Glucose needs to be added to specific levels to not interfere with seusequent islet usage.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Glucose</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td>G-7021&#160;</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:169px;">polysucrose and sodium diatrizoate solution</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td>Histopaque-10771</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Stereoscope</td>
			<td style="width:253px;">Carl-Zeiss, Oberkochen, Germany</td>
			<td>Stemi2000</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Stereoscope</td>
			<td style="width:253px;">Leica, Wetzlar, Germany</td>
			<td>Leica M165</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Microcentrifuge</td>
			<td style="width:253px;">Eppendorf, Hauppauge, NY</td>
			<td>Centrifuge 5417C</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Centrfuge</td>
			<td style="width:253px;">Eppendorf, Hauppauge, NY</td>
			<td>Centrifuge 5810R</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">15-ml centrfuge tubes&#160;</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>89039-666</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">50-ml centrfuge tubes&#160;</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>89039-658</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Precision balance</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>VWR-225AC</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Microfuge tubes</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>87003-294</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Pipetman P1000</td>
			<td style="width:253px;">Fisher Scientific,&#160; Waltham, MA</td>
			<td>F123602</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Pipetman P20</td>
			<td style="width:253px;">Fisher Scientific,&#160; Waltham, MA</td>
			<td>F123600</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">100X15 millimeter dish</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>25384-088</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">60X15 millimeter dish</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>25384-168</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">12-well plates</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>665-180</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">Scissor</td>
			<td style="width:253px;">Fine Scientific Tools, Foster City, CA</td>
			<td>14080-11</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Tweezers</td>
			<td style="width:253px;">Fine Scientific Tools, Foster City, CA</td>
			<td>5708-5</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">CD1 mice</td>
			<td style="width:253px;">Charles River Laboratories, Wilminton, MA&#160;</td>
			<td>CD-1</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">C57BL/6J</td>
			<td style="width:253px;">The Jackson laboratory, Farmington, CT&#160;</td>
			<td>C57BL/6J</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">B6CBAF1/J</td>
			<td style="width:253px;">The Jackson laboratory, Farmington, CT&#160;</td>
			<td>B6CBAF1/J</td>
			<td style="width:219px;"></td>
		</tr>
	</tbody>
</table>

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.

Under optimal conditions, the presented method can yield 20 - 80 islets from each small mouse pancreas. This number depends on the genetic background, age of mice, and the size of islets to be recovered. Among the commonly used, CD1 out-bred and C57BL/6J pure-bred mice produce less islets with smaller size than hybrids between CD1 and C57BL/6 or commercial B6CBAF1/J mice do. Direct hand picking generally gave a smaller number of islets, likely due to the exclusion of small islets that could be hard to recognized when mixed with exocrine tissues (Figure 1A-C). Gradient centrifugation can yield more islets, including smaller islets in the mix (Figure 1D). Older mice also produce more and larger islets, as expected from continued islet proliferation after birth. Interestingly, the islet number recovered is not directly correlated with pancreas size: CD1 mice usually have bigger pancreas than F1 progenies of CD1 and C57BL/6J mice crossing. Yet CD1 mice usually produce less islets than the F1 progenies.
Either insufficient- or over-digestion with collagenase results in suboptimal islet isolation. In the former case, large islets can be readily visualized, yet some islets cannot be completely separated from acinar tissues (Figure 2A). This will reduce the yield of islets, but larger islets are usually produced. In the latter case, acinar tissues can be completely dissociated from islets. Yet this compromises the islet structure, resulting in many islets with rough surfaces (Figure 2B).
Neonatal islets isolated by this method have expected insulin secretion profiles. For example, both P1 and P7 islets displayed high basal insulin secretion (Figure 3, compare the levels of insulin secretion at 2.8 mM glucose between P1-P7 and mature P24 islets), typical GSIS profiles of immature islet beta cells7, 9. This suggests that our islet isolation process largely conserves the functional properties of neonatal islets. We therefore expect that these islets are likely fit for other in vitro-based studies, including gene expression, metabolic analyses, survival assays, and stress responses.
<img alt="Figure 1" src="/files/ftp_upload/55160/55160fig1.jpg" /> 
Figure 1. Appearance of Islets during the Isolation Process. (A) P1 pancreata after collagenase digestion. Arrow points to a relatively large islet. Arrowheads point to two relatively small islets. (B) P1 islets after the first-round hand-picking. (C) Islets after the 3rd round handpicking. (D) Islet fractions after gradient centrifugation. Arrow, a large islet. Arrowhead, a small islet. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55160/55160fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55160/55160fig2.jpg" /> 
Figure 2. Islets after Insufficient- or Over- digestion with Collagenase. (A) Hand-picked islets after under-digestion, note the association between some islets (pink clusters, arrows) and acinar cells (dark clusters, arrowheads). (B) Islets after over-digestion, note the islets with jagged surfaces (arrows). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55160/55160fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55160/55160fig3.jpg" /> 
Figure 3. GSIS Results from Isolated Islets. Presented data are mean &#177; SEM. They represent the percentage of insulin release (amount of insulin released over the total amount of insulin contained in starting islets) within a 45 min assay window with indicated glucose concentration. Islets from ICR mice, via direct hand picking, were utilized for these assays. Note that P1 and P7 islets were considered immature, whereas P24 islets are mature7, 9. The P values are calculated between groups using t-test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55160/55160fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

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 ...

effective-isolation-of-functional-islets-from-neonatal-mouse-pancreas

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.0K Views.  Vanderbilt University Medical School. 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.

Video: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

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A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...