Lepr

The overall goal of this procedure is to obtain viable living eyelets from the diabetic LEP R DB mice, and assess insulin secretion from beta cells within the eyelets using live cell to photon imaging. This is accomplished by first preparing reagents such as the enzyme mixture for the pancreas digestion, the culture media for eyelet culturing, and the extracellular buffer for the two photon assay. Next, the abdomen of a euthanized mouse is opened up and the enzyme mixture is injected into the pancreas to digest the organ.

Then the eyelets are separated from the pancreatic digest and cultured. Finally, two photon microscopy is used to image single insulin granule fusions from beta cells within the eyelet. Ultimately, this protocol will help researchers to understand how insulin secretion is controlled in health and disease.

Main refinement of the eyelet isolation technique is the use of mixed enzymes to improve the yield and the viability of eyelets. The two photon imaging technique is currently the only method to record insulin secretion from multiple cells within an intact eyelet. To prepare enzymes to isolate pancreatic eyelets dilute one vial of five milligrams of liase TL with 26 milliliters of DM EM, prepare the collagenase type four in Hank's buffer to a concentration of 0.5 milligrams per milliliter, supplemented with the following mixed liase TL and collagenase solutions.

At a ratio four to one by volume aliquot the mixture of enzymes to 2.5 milliliters each and store at negative 20 degrees Celsius. To prepare RPMI isolation medium in a large conical flask containing one liter of water, dissolve one vial of RPMI 1 6 4 0 powder at room temperature and supplement with two grams of sodium bicarbonate and 4.02 grams of hippies. Use sodium hydroxide to adjust the pH to 7.4, then filter, sterilize, and store at four degrees Celsius.

Make RPMI culture medium by adding 10%FBS and 1%antibiotics to the RPMI isolation medium for two photon imaging. Prepare sodium rich extracellular buffer with the following reagents. To prepare extracellular dye, make eight millimolar stock of SRB an extracellular buffer aliquot into small tubes and store at negative 20 degrees Celsius.

To arrive at a working concentration, use fresh extracellular buffer to dilute the stock SRB one to 10. After sacrificing a mouse and using 70%ethanol to thoroughly spray the mouse body according to the text protocol, position the animal with the head toward and the tail away from the surgeon. Use two long lateral cuts through the abdominal skin and peritoneum to make a V-shaped flap.

Pull the flap of the skin up to uncover the entire abdominal cavity. Put aside the bowel and with the folded tissue, keep the liver in place to reveal the common bile duct. Next, apply a vascular clamp at the ambula on the duodenum wall to block the injected enzymes from entering the duodenum.

This is an important step that determines whether the pancreatic enzymes enter the pancreatic duct reflux, the injection site, or enter the duodenum. Use a 31 gauge needle to cannulate the common bile duct near the junction of the common hepatic duct and cystic duct and leave room for a second injection. If the pancreas fails to perfuse the first time, then slowly inject about two milliliters of cold enzyme mixture into the mouse common bile duct.

If the clamp is in the right position, the pancreas will be quickly perfused. Once the pancreas is perfused, quickly remove the organ, put it into a glass vial and incubate into 37 degrees Celsius water bath for approximately 19 minutes. After the incubation, stop the digestion process by adding approximately 20 milliliters of cold isolation medium.

Shake the vial gently to disrupt the pancreas and pour it through a normal T sive with a one millimeter pore size into a sterile 50 milliliter tube. Use the isolation medium to top off the tube and centrifuge at 400 G and four degrees Celsius for one minute. With the break off, add isolation medium to the palate and dissolve it.

Then transfer the suspension to two 15 milliliter tubes and spin a second time. After aspirating the supinate, add six milliliters of density gradient cell separation medium to each palette and pipette or vortex to resus. Then to the side of the tubes, gently add approximately six milliliters of isolation medium on top of the density gradient separation medium centrifuge at 100 G and four degrees Celsius for 15 minutes.

With the break off, collect the upper, middle and lower layers in three separate dishes. Then under a dissecting microscope, use a pipette to handpick the eyelets in the three dishes. The majority will be found in the middle layer to help the eyelets recover from the enzyme digestion.

Prepare groups of 40 eyelets in 25 centimeter Petri dishes with six milliliters of RPMI culture medium containing 10 millimolar glucose and culture for two days. Changing the medium daily to carry out two photon imaging as an indirect assay to measure insulin secretion. Begin by using three millimolar glucose in extracellular buffer to pre incubate two day cultured eyelets.

For 30 minutes, apply grease to the microscope stage to prevent the solution from leaking and mount thermo control chamber containing a glass slide on the microscope stage. Then with 500 microliters of extracellular buffer containing 0.8 millimolar, SRB and various glucose concentrations cover the chamber. Next, place the pre incubated eyelet into the middle of the chamber and focus at three to four cell layers into the eyelet.

Where in rodent eyelets, immuno staining for insulin shows that most cells are beta cells. Then with the two photon microscope and a 60 x oil immersion objective, identify single exo acidic events by the sudden appearance of a small fluorescent spot. Finally, in the scan image software confirm an event by the fast rising phase of the fluorescent signal over a region of interest for a wild type mouse.

About 200 eyelets are expected using the protocol demonstrated in this video. Healthy eyelets look bright, round shaped, and have a smooth border. And under digested isolation batch usually has small and fuzzy eyelets and asar cell attached eyelets.

While an over digested batch has fewer eyelets in the two photon assay in response to stimuli such as glucose or high potassium insulin granules fsed with the plasma membrane, an SRB enters the granules, which results in the appearance of a sudden fluorescent spot, approximately 400 nanometers in diameter. Within the time recorded all the exo events in response to the stimulus are identified and the location of the events, the time of the appearance, the duration and type of the fusion. Events like KISS and Run or full fusion are characterized and are valuable for assessing any defect in the granule fusion in models of type two diabetes.

This method was used to show that the defect in diabetic DB DB eyelets is the loss of full granule fusion and not a change in the kinetics of individual granule fusion. Furthermore, the biggest factor influencing decreased insulin secretion in disease is a reduction in the number of responsive cells. After watching this video, you should have a good understanding of the pancreatic perfusion and eye less actuation from the left, RDB mice, and also be able to perform the two for tongue essays to assess the incidences secretory responses within Intact isles.