Name: Murine Pancreatic Islet Isolation (Video) | JoVE Journal
Uploaded: 2007-08-22T00:00:00.000+00:00
Duration: 15 min 20 s
Description: Learn murine pancreatic islet isolation using collagenase digestion and Ficoll gradient purification. This method enables researchers to prepare viable islets for transplantation into diabetic NOD mice.

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Collagenase Preparation
<ol>
<li>Weigh out the collagenase into a 50 ml conical and add isolation buffer to make the final concentration, as indicated in the table below. Dissolve the collagenase by vortexing. (12 mice + 2 extra mice = 14 mice x 5 ml/mouse = 70 ml total collagenase solution) (70 ml x 0.5 mg/ml or 0.3 mg/ml = 35 mg or 21 mg of collagenase P total) 
<table style="BORDER-RIGHT: #ddd 1px solid; BORDER-TOP: #ddd 1px solid; BORDER-LEFT: #ddd 1px solid; BORDER-BOTTOM: #ddd 1px solid; TEXT-ALIGN: left" border="0" width="80%">
<tbody>
<tr>
<th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left">Strain</th><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left">Age</th><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left">Collagenase P (mg/ml)</th><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left">Time (min)</th>
</tr>
<tr>
<td style="font-size: 12px">C3H, Balb/C, B6</td>
<td style="font-size: 12px">&gt; 12 weeks</td>
<td style="font-size: 12px">0.8</td>
<td style="font-size: 12px">17</td>
</tr>
<tr>
<td style="font-size: 12px">C3H, Balb/C, B6</td>
<td style="font-size: 12px">&lt;= 12 weeks</td>
<td style="font-size: 12px">0.5</td>
<td style="font-size: 12px">13</td>
</tr>
<tr>
<td style="font-size: 12px">NOD, NOR, NON</td>
<td style="font-size: 12px">&gt; 12 weeks</td>
<td style="font-size: 12px">0.8</td>
<td style="font-size: 12px">17</td>
</tr>
<tr>
<td style="font-size: 12px">NOD, NOR, NON</td>
<td style="font-size: 12px">&lt;= 12 weeks</td>
<td style="font-size: 12px">0.5</td>
<td style="font-size: 12px">17</td>
</tr>
</tbody>
</table>
 </li>
<li>Dispense 2 ml of collagenase solution into each glass vial and place on ice. Load each 5 ml syringe with 3 ml of collagenase add 30g needle and place in ice (prepare in hood).</li>
</ol>
Distension
<ol>
<li>Just before dissection, sacrifice animal by cervical dislocation. Spray the whole body with 70% of ETOH, making it completely wet. Make a V-insision starting at the genital area. Rotate the mouse so the tail is facing away from you.</li>
<li>Remove the bowel to the left side of the open mouse. This will expose the pancreas and common bile duct.</li>
<li>Place a hemostat clamp on either side of the small-intestinal, where the bile duct drains, leaving a small pocket for collagenase solution to enter the intestine.</li>
<li>Inflate the pancreas through the bile duct with a 30g needle and 5 ml syringe containing 3 ml of cold collagenase solution, starting at the gall bladder.</li>
<li>Remove the pancreas from the body and place it in a siliconized vial containing 2 ml of collagenase solution. This step should be done QUICKLY and as cleanly as possible, minimizing collection of fat and connective tissue. </li>
<li>Place the vial on ice and repeat step 3 with the next mouse.</li>
</ol>
Digestion
<ol>
<li>Seal each vial and place it into a 37°C water-bath. Incubate for 13-17 minutes (time varies with strain and age of the animal).</li>
<li>After time has elapsed, shake the vial vigorously. The pancreas should fall apart.</li>
<li>Pour each digest through a large sterile sink strainer into a sterile 1000 ml beaker and forcefully pipette off the screen with washing buffer. Dispense the digests into 50 ml conicals tubes (3 pancreata/50 ml tube: if n=12, use enough washing buffer to make the final volume 200 ml and dispense it to 4x50 ml tubes). The dilution should be done rapidly and on ice to avoid unwanted islet digestion.</li>
<li>Spin down: Start the centrifuge. When it reaches 1300 rpm, turn it off.</li>
<li>Aspirate the supernatant, leaving ~5 mL. Be very cautious not to aspirate the pellet.</li>
<li>Resuspend pellet by tapping vigorously with your hand, then add 50 ml of wash buffer.</li>
<li>Spin down: Start the centrifuge. When it reaches 1300 rpm, turn it off.</li>
<li>Resuspend the pellet and wash with 5 ml of washing buffer.</li>
<li>Transfer the 5 ml to 15 ml conical and spin down, as above.</li>
<li>Aspirate the supernatant as completely as possible. (Remaining buffer might cause the change of the density of the Ficoll.)</li>
</ol>
Ficoll
Perform quickly. Long-term Ficoll exposure is TOXIC to islets.
<ol>
<li>Make sure pellet is completely broken up prior to adding ficoll (unbroken tissue is difficult to resuspend in ficoll).</li>
<li>Resuspend the pellet in 7 ml of ficoll, density 1.108, by vortexing vigorously.</li>
<li>Layer on top of each density of ficoll 2ml of each of the remaining densities in this order: 1.096, 1.069, then 1.037.</li>
<li>Spin for 15 minutes at 1800 rpm at 4 degrees, with break OFF!</li>
<li>Pick islets from the second layer using spoid (sterile plastic eyedropper). Transfer all collections to a 50 ml conical containing ~25 mL of cold buffer.</li>
<li>Wash, as above, 3 times with washing buffer (repeating step 12-15).</li>
<li>Resuspend Islets in 20 ml RPMI-1640 (containing 10% FCS and penicillin and streptomycin, HEPES, MEM-NEAA) and mix gently. Remove 100 ul of sample for counting.</li>
<li>Transfer 100 ul to 35 mm Grid-plate containing 1 ml of media and 1 ml dithizone.</li>
<li>Incubate remaining islets in RPMI 1640, in a 37°C, 7% CO2 incubator, in 160 mm plates with a total of 30 ml of media/plate. <img src="/files/ftp_upload/old/255_2008_8_24_3/255_Figure-01.png" alt="Density gradient centrifugation diagram, islet layer separation process, labeling 1.037-1.108." width="220" height="194" data-altupdatedat="1787630408000" data-alt="Figure 1"></li>
</ol>

<table><tbody><tr><td>Dithizone</td><td>reagent</td><td></td><td></td><td>Dissolve 5mg Dithizone in 5 ml of DMSO in a 50 ml conical tube. Add 45 ml of normal saline and mix gently. Filter with a 60 cc syringe and a 0.22 &quot;m syringe filter into a new 50 ml tube. Store at 4 OC, until use. Add 1 ml to each 35 mm counting Grid-plate, when needed.</td></tr><tr><td>PBS</td><td>Buffer</td><td></td><td></td><td></td></tr><tr><td>HBSS</td><td>Buffer</td><td></td><td></td><td></td></tr><tr><td>Isolation soln.</td><td>Buffer</td><td></td><td></td><td>HBSS containing 10 mM of HEPES, 1 mM of MgCl2, 5 mM of Glucose, pH 7.4</td></tr><tr><td>Wash soln.</td><td>Buffer</td><td></td><td></td><td>Isolation buffer containing 1mM CaCl2</td></tr><tr><td>Collagenase P</td><td></td><td>Boehringer Ingeheim</td><td>1213873</td><td></td></tr><tr><td>Dissection tools</td><td></td><td></td><td></td><td>Hemostat clamp (n=2), Forceps (n=2), Scissors (for skin incision an for taking out pancreas from adjacent tissue)</td></tr><tr><td>Syringe</td><td></td><td></td><td></td><td>sterile, 5ml, 1/mouse.</td></tr><tr><td>30 G needles</td><td></td><td></td><td></td><td>sterile, 1/syringe</td></tr><tr><td>siliconized glass vials w/Teflon cap</td><td></td><td>Fisher Scientific</td><td>213-018-54</td><td>sterile, 1/mouse</td></tr><tr><td>large sink strainer</td><td></td><td></td><td></td><td>sterile</td></tr><tr><td>1000 ml Beaker</td><td></td><td></td><td></td><td>sterile</td></tr><tr><td>Gauze pads</td><td></td><td></td><td></td><td>4x4, 2/mouse</td></tr><tr><td>plastic eye dropper</td><td></td><td></td><td></td><td>sterile</td></tr><tr><td>Mice</td><td>Animal</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

murine pancreatic islet isolation

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intest

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Murine Pancreatic Islet Isolation

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