The overall goal of this procedure is to successfully isolate eyelets from a mouse pancreas. This is accomplished by first isolating the pancreas from surrounding tissue. Next, the bile duct is cannulated so that the pancreas can be inflated with the collagenase protease mixture.
Once inflated, the pancreas is removed and the eyelets are carefully dissociated. Finally, the isolated eyelets are filtered and plated for future use. Ultimately, this method of eyelet isolation with purified collagenase and neutral protease is used to get maximal eyelet yield and optimal eyelet morphology.
The main advantage of this technique over existing methods of eyelet isolation is that we've eliminated the lot to lot variability by using purified enzymes. And in addition, we've changed or made improvements to some protocols to improve eyelet morphology. After euthanization, cover the torso with 70%ethanol before opening the abdominal cavity completely from anus to diaphragm.
Next place the mouse on the platform of a dissection microscope. Pull out the cecum and ascending colon and set them outside the body cavity to the left displace the lobes of the liver against the diaphragm. If they do not remain there on their own, increase the opening of the body cavity.
Grip the duodenum with curved forceps and locate the hepatic artery portal vein and bile duct bundle leading to the liver. With another pair of curved forceps. Insert a suture under the artery vein and duct bundle and tighten it as close to the liver as possible.
Using two pairs of curved forceps, carefully grab the duodenum and find the bile duct attached At the papilla, use one tong of the forceps to poke through the pancreas and connective tissue right under the bile duct and near the papilla working. Quickly, place the forceps through the hole and insert a suture.Loosely. Tie the suture around the bile duct to create a small circle using 90 degree forceps.
Pull the small intestine until the bile duct is tau. Make one horizontal cut across the papilla with super fine scissors without dislodging the bile duct from the papilla while holding the intestine. Insert a cannula into the bile duct and then gently pull the suture tight around the cannula.
Next, fill a three milliliter syringe with 2.0 milliliters of collagenase protease mixture. Inject this mixture into the cannula at a slow, constant pace. After the inflation of the pancreas is complete, remove the cannula from the bile duct.
Starting at the descending colon, snip the connective tissue while pulling the intestines up until the bile duct is reached. Next, separate the pancreas from the spleen and surrounding tissues. Finally, cut away the sutures and remove the pancreas.
Once removed, place the pancreas into a 50 milliliter tube containing five milliliters of HBSS on ice. Once all samples are collected, place any 50 milliliter tubes containing pancreas tissue into a 37 degree Celsius water bath. For 15 minutes, gently rock the tube and check for tissue dissociation.
Be sure the tissue comes apart easily holding the tube by the lid. Swirl 12 times to further dissociate the tissue. Add no more than 30 milliliters of HBSS and centrifuge.
Aspirate the supernatant carefully and leave a small amount of supernatant at the bottom so as to not disrupt the cell palette. Using a 30 milliliter syringe attached to 14 gauge needle, add no more than 10 milliliters of HBSS and aspirate the suspension up and down two times. Filter the cell mixture through a plastic T strainer into a 50 milliliter tube.
Rinse with another 10 milliliters, HBSS and centrifuge. Decant the supernatant and invert the tubes to drain on an absorbent paper towel for one minute. Resuspend each tube in 10 milliliters.
Cold 1100. His opaque overlay, the his opaque With 10 milliliters, HBSS and centrifuge. Remove the entire 20 milliliters of supernatant using a large bore 25 milliliter pipette and pass the supernatant through an inverted 70 micron filter.
Filter the eyelets directly into a Petri dish and rinse by pipetting 10 milliliters of eyelet media through the filter culture. The eyelets in a tissue culture incubator until ready for experimentation. Eyelet yields, morphology, and quality are the general parameters used to judge the success of eyelet isolation.
Typical eyelet morphology is shown here. The eyelets have a round to oblong shape with relatively uniform size.Here. Isolated eyelets were analyzed for glucose stimulated insulin secretion.
Insulin stimulation was similar between eyelets prepared by Sigma Type 11 as compared to ci zyme TM, MABP indicating a high quality of eyelet preparation. After watching this video, you should have a good understanding of how to cannulate a mouse bile duct. Work with zyme collagenase and neutro proteases to isolate your eyelids for any assay.
