Name: mouse islet of langerhans isolation using a combination of purified collagenase and neutral protease
Uploaded: 2012-09-07T13:00:00
Duration: 6 min 59 s
Description: Watch this Scientific Journal Video about Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease at JoVE.com

This work was supported in part by NIH grants R01 DK060581, R01 DK083583 (both to RGM).

A. Materials Needed: See Table 1 (reagents) prior to preparation
<ol>
 <li>HBSS(P/S): 100 ml 10X HBSS + 900 ml H20 + 1% Penicillin/Streptomycin (stored in the refrigerator or kept on ice). </li>
 <li>20% BSA stock is made in water and aliquoted and stored at -20 &deg;C.</li>
 <li>HBSS(BSA): 200 ml 1X HBSS(P/S) + 3 ml of 20% BSA (final BSA concentrations is 0.3%) </li>
 <li>Islet medium: RPMI + 10% FBS + 1% glutamine + 1% Penicillin/Streptomycin </li>
 <li>Histopaque 1100: 240 ml 1119 Histopaque + 200 ml 1077 Histopaque </li>
 <li>4-0 Suture (McKesson): cut into 2 &frac34; inch pieces and stored in a 10 cm Petri dish </li>
 <li>Instruments: Bend the forceps from Fine Science Tools to 90 &deg;, and file down the serrated teeth of all three pairs of the forceps. The goal is to dull the teeth, not to remove them. </li>
 <li>Cannula for administration of collagenase/protease mixture: 27G &frac12; in. needle, 30 &frac12; in. needle, PE50 tubing cut into 12 in. pieces. File both needles down to a smooth rounded end that has no sharp edges. Insert the 27G needle into one end of a 12 in. piece of PE50 tubing. Remove the 30G needle from the casing by smashing the casing with a pair of pliers, turning the casing &frac14; turn each time. Remove the needle with the pliers from the casing and insert it into the other end of the PE50 tubing under a dissection microscope. Be sure to not kink the tubing as you insert the needle into the PE50 tubing. The 27G needle is the end that you will attach the syringe of collagenase. </li>
 <li>Collagenase (CIzyme MA) and Neutral Protease (CIzyme BP). Reconstitute the TDE per manufacturer's instructions. Refer to the lot specific Certificate of Analysis to calculate the enzyme activity concentration. Transfer a total of 350,000 collagen degrading activity (CDA) units of the reconstituted collagenase and 100,000 neutral protease units of the reconstituted BP Protease into a conical tube and dilute up to 15 ml total in HBSS (P/S) </li>
</ol>
B. Step-by-Step Protocol
1. Inflation of Pancreas with Collagenase/Protease Mixture
<ol>
 <li>Euthanize mouse by cervical dislocation, saturate the mouse torso with 70% EtOH, open abdominal cavity completely from anus to diaphragm with any dissection scissors and forceps.</li>
 <li>Place the mouse on the platform of a dissection microscope. </li>
 <li>Pull the caecum and ascending colon out and set them outside the body cavity to your left. See Figure 1 for a summary of the following steps. </li>
 <li>Position the lobes of the liver against the diaphragm; they should remain there if the body cavity is open far enough. Find the hepatic artery, portal vein and bile duct bundle leading into the liver by gripping the duodenum with curved forceps. With another pair of curved forceps, reach under the artery, vein, and duct bundle and pull a 2&frac34; in. piece of 4-0 suture under the artery, vein, bile duct and tie it once and pull it tight as close to the liver as possible.</li>
 <li>Using two pairs of curved forceps, carefully grab the duodenum and find the bile duct attached to the duodenum at the papilla. Use a tong of the forceps to poke through the pancreas and connective tissue right under the bile duct close to the papilla. Poke your forceps quickly through the organ to make a hole and then put both tongs of forceps through and grab another 2&frac34; inch piece of 4-0 suture through and tie it once and pull it to a small circle, but do NOT pull tight. </li>
 <li>Change to the 90 &deg; forceps and to the Superfine Vannas scissors. With the 90 &deg; forceps, carefully pinch and pull the small intestine until the bile duct is taut. Make one horizontal cut across the papilla with the scissors by stabbing the open scissors into the intestine and then cutting with one motion. Try to only make one cut across the papilla without dislodging the bile duct from the papilla. </li>
 <li>While still holding the intestine with the 90 &deg; forceps, insert the cannula into the bile duct up to half the length of the bile duct, and then gently pull the suture tight around the cannula. </li>
 <li>Fill a 3 ml syringe with 2.0 ml of collagenase/protease mixture and inject into the cannula at a slow, constant pace. </li>
 <li>After inflation of the pancreas is complete, remove cannula from bile duct and use the curved forceps and curved spring scissors to remove the inflated pancreas by starting at the descending colon and snipping the connective tissue as you pull the intestines up. Continue to remove the pancreas from the intestines until you reach the bile duct, then remove the pancreas from the spleen, then the stomach and then from the viscera of the abdominal cavity. Finish removing by snipping away the sutures. 
 Add the pancreas to a 50 ml tube containing 5 ml of HBSS(P/S). This tube should be on ice. </li>
 <li>Repeat the procedure above for each animal up to four animals. For optimal results, dissociate only four pancreata at one time. Typically, while the first four are in the 37 &deg;C water bath, we inflate another four pancreata. </li>
</ol>
2. Pancreas Dissociation
<ol>
 <li>Place the 50 ml tubes containing the pancreata into a 37 &deg;C water bath for 15 min. </li>
 <li>Gently rock the tube and check for tissue dissociation, be sure tissue comes apart easily, then swirl the tubes 12 times while you are holding the tube by the lid.</li>
 <li>Add no more than 30 ml of HBSS(BSA) (this can be a lower amount, depending on how much is required to balance the tubes for the centrifugation step), and centrifuge at 290 x g for one min.</li>
 <li>Aspirate supernatant carefully and leave a small amount of supernatant (2-3 ml) at the bottom so as to not disrupt the cell pellet. </li>
 <li>Using a 30 ml syringe attached to 14G needle, add no more than 10 ml of HBSS(BSA) and aspirate the suspension up and down 2 times.</li>
 <li>Filter the cell mixture through a plastic tea strainer into a 50 ml tube and rinse with another 10 ml HBSS(BSA).</li>
 <li>Centrifuge at 330 x g for 2 min.</li>
 <li>Decant supernatant and invert tubes to drain on an absorbent paper towel for 1 min.</li>
 <li>Resuspend each tube in 10 ml cold 1100 histopaque.</li>
 <li>Overlay the histopaque with 10 ml HBSS(BSA) and centrifuge at 900 x g for 18 min.</li>
 <li>Remove the entire 20 ml of supernatant using a large bore 25 ml pipet and pass the supernatant through an inverted 70 &mu;m filter. </li>
 <li>Rinse the islets into a 10 cm Petri dish by pipetting 10 ml islet media through the filter while holding it right side up over the dish. </li>
 <li>Culture the islets in a 37 &deg;C, 5% CO2 tissue culture incubator until ready for experimentation. Typically, islets are hand-picked and counted prior to experimentation. </li>
</ol>
3. Representative Results
Islet yields, morphology, and quality are the general parameters used to judge the success of islet isolation. In our hands, islet yields from the average C57BL/6 mouse are in the range of 150-250 islets using this protocol (see Table 2), and are comparable to data obtained using optimized Sigma type XI collagenase. However, yields can vary depending upon the mouse strain used and the age of the mouse. Most of the animals we employ are in the 8-16 week age range This protocol has been tested on multiple mouse strains, including C57BL/6 (180-250 islets/mouse), CD1 (200-300 islets/mouse), 129/B6 (200-300 islets/mouse), BLKS-db/db (120-200 islets/mouse), NOD (10 week-old, 100-150 islets/mouse), and NOD-SCID (100-150 islets/mouse). Typical islet morphology is shown in Figure 2, in which islets have a round to oblong shape with relatively uniform size (although size uniformity can vary from strain to strain). Figure 3 shows our typical analysis of glucose-stimulated insulin secretion from C57BL/6 mouse islets isolated using purified TDEs vs. Sigma type XI collagenase. Typically, a high quality islet preparation results in an insulin stimulation at 25 mM glucose that is 6-20 times greater than that observed at 2.5 mM glucose. Other applications can also be used to test the quality of isolated islets, and these include use of perifusion techniques, Ca2+ imaging (with Fura2), and reverse-transcription PCR, among others11.
<img src="/files/ftp_upload/4137/4137fig1.jpg" alt="Figure 1" /> 
Figure 1. A summary of the cannulation of the bile duct. 
<img src="/files/ftp_upload/4137/4137fig2.jpg" alt="Figure 2" /> 
Figure 2. Typical appearance of C57BL/6 islets at 24 hr following isolation. Images were acquired on an inverted microscope at 40X magnification.
<img src="/files/ftp_upload/4137/4137fig3.jpg" alt="Figure 3" /> 
Figure 3. Glucose-stimulated insulin secretion of C57BL/6 islets. At 24 hr following isolation, 100 islets were incubated in a 12-well dish for 1 hr at 37 &deg;C in Krebs-Ringer HEPES-buffered solution containing 2.5 mM glucose. Islets were then transferred to Krebs-Ringer HEPES at 2.5 mM glucose for an additional hr, after which the supernatant was collected for insulin measurement using a two-site immunospecific enzyme-linked immunosorbent assay (ELISA) (Crystal Chem). The same islets were subsequently transferred to Krebs-Ringer HEPES-buffered solution containing 25 mM glucose, and insulin release into the medium was measured by ELISA after 1 hr of incubation.

<ol>
	<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> Nat. Protoc. 4, 1649-1652 (2009).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+pancreatic+islet+isolation.">Murine pancreatic islet isolation.</a> J. Vis. Exp. (7), e255 (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), e2096 (2011).</li><li>Kin, T., O'Gorman, D., Senior, P., Shapiro, A. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience+of+islet+isolation+without+neutral+protease+supplementation.">Experience of islet isolation without neutral protease supplementation.</a> Islets. 2, 278-282 (2010).</li><li>Gramignoli, R., Green, M. L., Tahan, V., Dorko, K., Skvorak, K. J., Marongiu, F., Zao, W., Venkataramanan, R., Ellis, E. C. S., Geller, D., Breite, A. G., Dwulet, F. E., McCarthy, R. C., Strom, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+purified+tissue+dissociation+enzyme+mixtures+for+human+hepatocyte+isolation.">Development and application of purified tissue dissociation enzyme mixtures for human hepatocyte isolation.</a> Cell Transplant. , (2012).</li><li>McCarthy, R. C., Spurlin, B., Wright, M. J., Breite, A. G., Sturdevant, L. K., Dwulet, C. S., Dwulet, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+collagen+degradation+assay+to+assess+purified+collagenase+used+in+islet+isolation.">Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation.</a> Transplant. Proc. 40, 339-342 (2008).</li><li>Gotoh, M., Ohzato, H., Porter, J., Maki, T., Monaco, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+role+of+pancreatic+ductal+collagenase+injection+for+isolation+of+pancreatic+islets.">Crucial role of pancreatic ductal collagenase injection for isolation of pancreatic islets.</a> Horm. Metab. Res. Suppl. 25, 10-16 (1990).</li><li>Ohzato, H., Gotoh, M., Monden, M., Dono, K., Kanai, T., Mori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+in+islet+yield+from+a+cold-preserved+pancreas+by+pancreatic+ductal+collagenase+distention+at+the+time+of+harvesting.">Improvement in islet yield from a cold-preserved pancreas by pancreatic ductal collagenase distention at the time of harvesting.</a> Transplantation. 51, 566-570 (1991).</li><li>McCall, M. D., Maciver, A. H., Pawlick, R., Edgar, R., Shapiro, A. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopaque+provides+optimal+mouse+islet+purification+kinetics:+comparison+study+with+Ficoll,+iodixanol+and+dextran.">Histopaque provides optimal mouse islet purification kinetics: comparison study with Ficoll, iodixanol and dextran.</a> Islets. 3, 144-149 (2011).</li><li>Breite, A. G., Dwulet, F. E., McCarthy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+dissociation+enzyme+neutral+protease+assessment.">Tissue dissociation enzyme neutral protease assessment.</a> Transplant. Proc. 42, 2052-2054 (2010).</li><li>Evans-Molina, C., Robbins, R. D., Kono, T., Tersey, S. A., Vestermark, G. L., Nunemaker, C. S., Garmey, J. C., Deering, T. G., Keller, S. R., Maier, B., 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=PPAR-{gamma}+Activation+Restores+Islet+Function+in+Diabetic+Mice+Through+Reduction+of+ER+Stress+and+Maintenance+of+Euchromatin+Structure.">PPAR-{gamma} Activation Restores Islet Function in Diabetic Mice Through Reduction of ER Stress and Maintenance of Euchromatin Structure.</a> Mol. Cell. Biol. 29, 2053-2067 (2009).</li></ol>

NDS, SAT, and RGM have nothing to disclose. Author AGB is employed by VitaCyte. Author RCM has an ownership interest in VitaCyte.

In this protocol, we have described our procedure for the cannulation, collagenase perfusion, and dissociation of the murine pancreas, with the goal of isolating the islets of Langerhans. Technically, our method, once mastered, should allow for pancreas isolation within 4 min of euthanizing the animal, and by 1 hr should result in the isolation of islets from a single animal. The rapidity of the technique allows us to isolate islets from up to 12 mice at a typical stretch, thereby resulting in the isolation of up to 3,000 islets. 
Unlike other protocols that use crude preparations of collagenase, our protocol features the use of purified proteases, CIzyme Collagenase MA and CIzyme BP Protease. Variations in the consistency of TDE preparations that are commercially available requires that each lot be individually tested and optimized prior to general use. This lot-to-lot variability led us to consider use of purified TDEs from VitaCyte. These highly purified TDEs are characterized by multiple methods including the use of sensitive fluorescence labeled substrate assays. The fluorescence CDA assay is more sensitive to different molecular forms of collagenase that have different kinetics in degrading native collagen. Historical peptide substrate assays provide an incomplete assessment of collagenase. Characterizing collagenase by multiple methods ensures greater enzyme activity between lots. 
Based on our in-house testing using a variety of collagenase preparations commercially available, we found that the purified enzyme combinations yielded reproducible lot-to-lot results. The major advantages of using highly purified and rigorously characterized TDEs in this application are once the optimal enzyme composition is defined, the lot-to-lot performance consistency is assured. This consistency eliminates the labor intensive process of lot qualification normally required for crude or enriched collagenase products. Purified TDEs also enable additional modifications of the composition to improve the yield and quality of islets recovered from different mouse strains. Reporting optimal enzyme compositions for isolation of islets from different strains of mice can be more effectively communicated. This, in turn, leads to more productive use of resources and improved flexibility in experimental design. 
There are many critical steps that can affect the outcome of islet isolations using our protocol. The first is the need to achieve effective inflation of the pancreas during the perfusion of the enzyme preparation. It is important to start inflation with gentle force on the syringe, and increase the force as the pancreas inflates. It is helpful to move the intestines and lift the pancreas during inflation so that the collagenase perfuses the entire organ. Another critical step is the force with which one shakes the tubes after incubation at 37 &deg;C (step 2.2). We have found that shaking the pancreas hard against the cap of the tube causes fragmentation of islets, but without any form of mechanical dissociation at this step, islet yields can fall dramatically. It is also imperative that during the dissociation step with the syringe (step 2.5), a medium-level force is applied during the up and down motion with the plunger; this ensures adequate tissue dissociation before the filtering step (2.6), where very little tissue should be retained on the tea strainer filter. Finally, the last step to follow carefully is the aspiration of the entire 20 ml of the histopaque fractionated islets. Although islets are typically at the interface of the histopaque and HBSS, we have found that we lose islets when we try to remove only the interface; instead, we now remove the entire 20 ml using a large bore 25 ml pipet. We added the filtration through the inverted 70 &mu;m filter (step 2.11) to eliminate the need to centrifuge the islets repeatedly to wash away the histopaque.

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>&nbsp;</td>
 <td>Catalogue #</td>
 </tr>
 <tr>
 <td>10X HBSS</td>
 <td>Life Technologies</td>
 <td>14065</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Fisher Scientific</td>
 <td>10-040</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma Aldrich</td>
 <td>any</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Histopaque 1077</td>
 <td>Sigma Aldrich</td>
 <td>10771</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Histopaque 1119</td>
 <td> Sigma Aldrich</td>
 <td>11191</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>4-0 Suture 100 yd roll</td>
 <td>McKesson </td>
 <td>451521</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>14 g blunt end needle</td>
 <td> Fisher Scientific</td>
 <td>14-8216M</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Vannas Superfine Sciss</td>
 <td>Fisher Scientific</td>
 <td>501778</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Curved Spring Scissors</td>
 <td> Fisher Scientific</td>
 <td>14127</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Curved Forceps (2)</td>
 <td>Fisher Scientific</td>
 <td>15914G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Curved forceps (for 90 &deg;)</td>
 <td> Fine Science Tools</td>
 <td>11052-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>27G 1/2 inch needle</td>
 <td>Fisher Scientific</td>
 <td>305109</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>30G 1/2 inch needle</td>
 <td>Fisher Scientific</td>
 <td>305106</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PE tubing</td>
 <td>Becton Dickinson</td>
 <td>427411</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Collagenase MA</td>
 <td>Vitacyte, LLC</td>
 <td>001-2030</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BP Protease</td>
 <td> Vitacyte, LLC</td>
 <td>003-1000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3 ml syringe</td>
 <td>Fisher Scientific</td>
 <td>309585</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>30 ml syringe</td>
 <td>Fisher Scientific</td>
 <td>309650</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>70 &mu;m filter</td>
 <td> Fisher Scientific</td>
 <td> 352350</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 cm2 Petri Dish</td>
 <td>Fisher Scientific</td>
 <td>351005</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Plastic tea strainer</td>
 <td>Any kitchen supply</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td></td>
 <td>Table 1. Reagents and Equipment</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td></td>
 <td><table border="1" cellspacing="0" cellpadding="0" width="510">
 <tbody>
 <tr>
 <td>Sigma type XI 
 (l010M8618)</td>
 <td>MA (100615) 
 BP (110329)</td>
 <td>MA (081104) 
 BP (100823)</td>
 <td>MA (091211) 
 BP (101109)</td>
 </tr>
 <tr>
 <td>216 (&plusmn;74)</td>
 <td>260 (&plusmn;45)</td>
 <td> 157 (&plusmn;37)</td>
 <td>200 (&plusmn;4)</td>
 </tr>
 </tbody>
 </table></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td></td>
 <td>Table 2. Average islet yields (&plusmn;SD) using different TDE lots*
 *Data represent average islet yields from at least 10 mice. Lot numbers in parentheses</td>
 </tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease at JoVE.com

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

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 ...

mouse-islet-langerhans-isolation-using-combination-purified

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Biology

Murine Pancreatic Islet Isolation

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.

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...