The overall goal of this procedure is to isolate healthy eyelets for measuring differences in cyclic A MP production in response to various experimental treatments. This is accomplished by first inflating and then removing the pancreas in the second step. The healthy eyelets are isolated through a series of spins and washes.
Next, the viable eyelets are picked into fresh islet culture media and allowed to incubate overnight. In the final step, the eyelets are exposed to the desired experimental treatments and a cyclic A MP assay is performed. Ultimately, an enzyme immunoassay is used to measure changes in cyclic A MP production in response to different treatments applied to the eyelets.
Generally, people new to this method will struggle because it takes a lot of practice to achieve a full pancreatic inflation. First preload a syringe and cannula with five milliliters of collagenase solution. Load a blunted 30 gauge needle onto the cannula and place the syringe in cannula on ice.
After moving the small intestine to the right side of the mouse, find the sphincter of od, which is at the end of the common bile duct and splays out onto the small intestine, forming a white triangle. Once the sphincter has been located, take a pair of number five dumont forceps and slide the tip underneath the common bile duct as close to the small intestine as possible. Being careful not to pierce the duct.
After piercing through the small intestine and connective tissue, use the tip of the dumont forceps to grasp the suture and pull it under the common bile duct. Tie off the common bile duct as close to the sphincter of OD as possible, making sure the knot is taught to prevent leakage. Next, grasp both ends of the suture and pull it towards the tail to put some tension on the common bile duct.
Then using a free hand, make a small incision with a pair of micro scissors in the common bile duct just above the last bifurcation into the liver holding the two ends of the string with the common bile duct taut. Remove the syringe and cannula from the ice bucket. Set down the syringe and insert the prepared blunted 30 gauge needle into the cut.
Common bile duct. Being careful not to pierce through the wall of the duct. Obtaining a full pancreas inflation is the trickiest part.
We use a needle that gives a good seal. If the first needle doesn't give a good seal, use a larger one, like a 27 gauge needle. Now depress the plunger of the syringe slowly letting the pressure off in a pulsatile fashion until the first part of the pancreas inflates.
Follow this with the gentle constant pressure until the pancreas no longer inflates. A successful inflation will have an even distribution of exocrine tissue, and the part of the pancreas on top of the stomach will be inflated. To remove the pancreas, use a pair of forceps in one hand to grasp the small intestine at the sphincter of od.
With the other hand, insert the closed tip of a curved pair of scissors to separate part of the pancreas from the small intestine. Move the tip down the small intestine to the pink connective tissue. Then pick up the small intestine near where it attaches to the stomach and cut the pancreas away from the rest of the small intestine.
Grab as much of the pancreas as possible and lift it gently. With the curved scissors. Cut away the remaining connections between the pancreas and the thoracic cavity.
To fully remove the pancreas store each removed pancreas in about 2.5 milliliters of collagenase solution. An individual 50 milliliter conical tubes on ice to wash the pancreas's. First, transfer each pancreas to a new 50 milliliter conical tube containing 25 milliliters of collagenase.
Then place the pancreas containing 50 milliliter conical tubes. Upright in a 37 degrees Celsius shaking water bath, capable of oscillating at 220 to 250 RPM with the shaker turned off. Now gas each 50 milliliter conical tube with 95%oxygen and 5%carbon dioxide for five minutes.
With a pasture pipette, recap each tube tightly and place them sideways in the shaking water bath underneath the surface of the water. Starting at minute six, shake the tubes at 220 to 250 RPM for 20 seconds every other minute for up to 16 minutes. After shaking, spin the tubes at 400 to 500 Gs at room temperature immediately turning the centrifuge off once it has reached full speed.
Next, without disturbing the pellet, use a vacuum trap to aspirate about 22.5 milliliters of the collagenase solution from each tube. Then resuspend the pellets in 12.5 milliliter of HBSS and vortex the tubes gently to break up the pellets. After washing and vortexing the cells and more HBSS, pour each cell suspension through a 1000 micron mesh into new individual 50 milliliter conical tubes.
Then after spinning down the cell suspensions again, use a vacuum trap to carefully aspirate all the HBSS without disturbing the pellet. Once all of the HBSS has been removed, add 4.8 milliliters of 25%F call solution to each tube. After vortexing to break up the pellet, carefully dispense 2.4 milliliters of 23%20.5%and 11%fial solution along the side of the tube rotating the 50 milliliter conical tube.
To ensure even layering after centrifuging the FI call solution, pour all the FI call into a fresh 50 milliliter conical tube, taking care to leave the pellet of exocrine tissue behind. Add ice cold HBSS to the new tube up to the 50 milliliter mark. Then cap and invert the tube four to six times.
To mix the FI call and HBSS together. Use the vacuum trap to carefully aspirate most of the HBSS fial mixture without disturbing the loose pallette. Then transfer the palate to a new tube.
Use a pasture pipette to transfer the eyelets into a Petri dish containing eyelet culture. Media taking care to avoid transferring ASIN art tissue debris. Place the Petri dish onto a dissecting microscope with a backlight.
Healthy eyelets will be spherical in shape and have a golden brown to dark brown center. Any eyelets connected to ASIN or tissue, which will appear dull and gray, should not be used and should be discarded. Moreover, larger eyelets which develop a dark necrotic center after an overnight incubation should be discarded as they do not function properly.
If the eyelet preparation contains a large amount of debris, handpick the eyelets twice into fresh medium, add DNA to the digestion buffer to help to limit eyelet clumping. Begin this step by swirling the eyelets to the middle of the Petri dish. Using a P 20 pipette, transfer the eyelets to a new 15 millimeter by 60 millimeter polystyrene Petri dish containing five milliliters of the pre incubation solution to wash the glucose containing culture medium from the eyelets.
Then use the P 20 pipette to transfer at least 13 eyelets into individual micro fuge tubes containing one milliliter of freshly gassed Krebs ringer bicarbonate buffer, maintaining eyelet size consistency between the tubes. After incubating the samples at 37 degrees Celsius and 5%CO2 for 45 minutes cap, quickly vortex and spin the fuge tubes in a tabletop centrifuge at room temperature up to 7, 000 to 7, 500 Gs.If insulin or another metabolite is a desired downstream application, use a P 1000 pipette to collect an aliquot of medium in a micro fuge tube. If stimulation media is not being collected, it may be discarded.
Use a P 1000 pipette to remove most of the Krebs in each micro fuge tube, leaving approximately 10 to 15 microliters of Krebs on the eyelet pellet. Then use a P 20 pipette to remove the portion closest to the pellet. Finally, once there is less than 10 to 15 microliters of crebs left on the eyelet snap freeze the eyelet palettes in liquid nitrogen.
Then store the snap frozen pellets at minus 80 degrees Celsius until the cyclic A MP is measured. Typically, the impact of an agent on cyclic a MP production directly correlates with its impact on insulin secretion. In this experiment, isolated eyelets from Wildtype or gene knockout mice were stimulated with 11.1 millimolar glucose and intracellular cyclic.
A MP production was measured and normalized to the total cellular protein. Secreted insulin was measured using a standard insulin, Eliza, and also normalized to total cellular protein. As expected, the change in cyclic A MP production correlated directly with the augmentation in glucose stimulated insulin secretion Petrol.
After watching this video, you should be able to isolate culture and measure cyclic a and p levels in healthy mouse eyelets.
