A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Podsumowanie

Assaying in vitro β-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining β-cell function.

Streszczenie

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 β-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 β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-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, [3^H]-thymidine incorporation, protein abundance, and mRNA expression.

Wprowadzenie

The strict maintenance of euglycemia is imperative to prevent morbidities such as neuropathy, nephropathy, and retinopathy, which are all hallmarks of the pathology of uncontrolled type 1 and 2 diabetes¹. Reduced β-cell function and mass in both type 1 and 2 diabetes perturb blood glucose concentrations². Whereas immune-mediated type 1 diabetes results from a devastating loss of insulin-producing β-cells, impaired β-cell insulin secretion and peripheral insulin signaling in type 2 diabetes together promote hyperglycemia, dyslipidemia, and increased hepatic glucose production, which eventually results in both loss of β-cell mass and insulin secretory capacity from individual β-cells³. Understanding the underlying β-cell mechanisms in the progression of type 1 and 2 diabetes will hopefully give rise to novel therapies to prevent and treat these diseases.

In vitro tissue culture models, such as the INS-1 and MIN6 immortalized β-cell lines, can be useful tools for understanding specific β-cell functions. However, the interactions among the different cell types within the islet may themselves regulate β-cell function. For example, the paracrine influence of glucagon (released from α-cells) and somatostatin (released from δ-cells) in increasing and decreasing insulin secretion, respectively, demonstrates the importance of cell cell proximity in the endocrine response⁴. Moreover, gap junctions between β-cells potentiate the release of insulin⁵. Furthermore, although strides have been made in generating insulinoma lines that better replicated the physiological response of isolated islets to glucose (e.g., the INS-1- derived 832/13 and 832/3 cell lines), their glucose responsiveness still differs from normal rat islets^6,7. Moreover, the response of these clonal insulinoma cell lines to glucagon-like peptide-1 (GLP-1) agonists can differ dramatically from one another, as well as from normal islets⁶. Therefore, immortalized cell lines may not represent the best model for assaying agents that impact on cAMP production.

In contrast to the insulinoma-derived cell lines, studying β-cell function solely in whole animal models offers its own set of complications. One of the biggest challenges in working with endocrine tissue is measuring the precise concentration of hormone released. Specifically, the liver plays a major role metabolizing insulin, and the pancreas blood flow goes directly to the liver. Thus, a plasma insulin measurement may not accurately portray the amounts of insulin being secreted from the pancreas itself or the impact of different treatments on the rate of insulin secretion⁸. Furthermore, renal metabolism of glucagon may limit the reliability of glucagon output from islet α-cells⁹. Therefore, isolating primary mouse islets for in vitro experimentation provides a more precise understanding of how the islet is responding to specific stimuli to complement measurements made in vivo.

The present protocol for the isolation of mouse islets is a well-established protocol used by a number of groups (with slight modifications that may help to increase success)^10,11. In addition, the determination of cAMP production allows for a direct read-out of the incretin responsiveness of the β-cells. In conjunction with cAMP measurement, protein content and insulin secretion can also be quantified from the same cAMP sample prep, helping to determine whether a defect in β-cell function lies proximal or distal to cAMP¹⁰. The final cAMP content and insulin secretion application in this protocol can be a very powerful tool for understanding the influence of pharmaceutical and dietary constituents, among others, on cAMP and insulin secretion. In addition to stimulation from glucose alone, other compounds can be used to measure changes in cAMP and insulin secretion^10,11.

Finally, although insulin is the primary hormone we assay from isolated islets, other hormones, such as glucagon and somatostatin, as well as cytokines, eicosanoids, and cyclic adenosine monophosphate, can also be measured, either by a transient stimulation assay or by quantification of their levels in culture medium¹². Finally, although outside of the scope of this manuscript, islet isolation with the described collagenase isolation method allows for islet preservation so that many other downstream applications may be pursued, such as islet transplantation, RNA isolation for quantitative real time PCR or microarray analyses, protein isolation for Western blotting, islet embedding and immunofluorescent imaging, and [3H]-thymidine incorporation as a measure of islet cell replication, some of which have been described in previous JoVE articles^13-16. Overall, following the islet isolation procedure described in the protocol may provide a researcher with important and useful information for developing therapies and promoting drug discovery aimed at enhancing β-cell function.

Protokół

All animal experiments were executed in compliance with all relevant guidelines, regulations and regulatory agencies. The protocol being demonstrated was performed under the guidance and approval of the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin-Madison.

1. Preparation of Solutions

1. The method of euthanasia in this protocol is exsanguination under Avertin anesthesia (for a review of alternatives, see Discussion). To make Avertin, add 0.625 g (1.25% or 44.2 mM) of 2-2-2 tribromoethanol to 1.25 ml of 2-methyl-2-butanol in a 15 ml conical tube and heat at 37 °C for 20-30 min. Vortex the solution on high for 10-15 sec at a time to fully dissolve the 2-2-2 tribromoethanol. Once fully dissolved, add the solution to 48.75 ml of double distilled H₂O, filter through a 0.45 μm filter into a new 50 ml conical tube, wrap the 50 ml conical tube in aluminum foil and store at 4 °C for up to one month. Bring the tube to RT before injecting mice.
2. To make Hanks’ Balanced Salt Solution (HBSS), make up 1 L 1x HBSS from a 10x stock (Gibco, Life Technologies, Carlsbad, CA) in double distilled H₂O, add 0.35 g (4.2 mM) NaCHO₃, and store at 4 °C for up to 1 month. For each mouse, 105 ml of this solution (plus 5-10 ml extra for pipetting error) will be required for islet isolation.
3. Islets are very susceptible to hypoxic damage; therefore, HBSS is bubbled with 95% O₂/ 5% CO₂ to mimic the concentrations of O₂ in the blood. Special gas mixes may be purchased and a bubbling station set up with a Pasteur pipette attached to flexible tubing. After bubbling the required amount of HBSS with 95% O₂/ 5% CO₂ for 15 min, add 0.02% RIA grade BSA (bovine serum albumin) by volume and keep on ice for the remainder of the islet isolation.
4. For the Ficoll preparation, weigh out 32.5 g of Ficoll in a 400 ml beaker, add 80 ml of HBSS, and stir at 500-700 rpm for 30 min. Once dissolved, pour the Ficoll solution into a 250 ml graduated cylinder and add HBSS to the 130 ml mark on the graduated cylinder to create a 25% Ficoll solution. Cover the top of the graduated cylinder with two portions of Parafilm® M (Pechiney Plastic Packaging Company, Chicago, IL) and shake vigorously several times.
5. For a 23%, 20.5%, and 11% Ficoll solution, add 27.6, 24.6, and 13.2 ml of the 25% Ficoll, respectively, to 50 ml conical tubes. Then, bring each solution up to a total of 30 ml with HBSS and shake well to mix. All four Ficoll solutions should be stored at 4 °C and are good for up to 2 weeks.
6. Collagenase solution that is suitable for isolating active islets is prepared from collagenase isolated from Clostridium histolyticum (Materials table). Each lot needs to be tested for enzyme efficiency. Typically, collagenase is used at 0.3-0.5 mg per ml of HBSS. Utilizing enzyme concentrations in that range (usually 3 different concentrations), determine the quality and quantity of isolated islets from age-matched mice or littermates to set the working enzyme concentration.
7. Once the correct concentration is determined, each mouse requires 35 ml of collagenase in HBSS for the entire isolation protocol. Swirl the collagenase in HBSS to dissolve. Immediately prior to the surgery, pre-load a 5 ml syringe with collagenase solution, place the cannula on and remove air bubbles, and leave on ice until needed.
8. The islet culture media for O/N incubation is a supplemented RPMI 1640 media. For the stock solution, dissolve one RPMI 1640 packet (Gibco, New York) in 1 L of double distilled H₂O and add 1.19 g HEPES (5 mM) and 2 g NaCHO₃ (24 mM). Adjust the pH to 7.4, filter sterilize, and store at 4 °C in the dark.
9. For the supplemented media, add 10% FBS and 100 IU/100 μg/ml penicillin/streptomycin, respectively, to the stock RPMI 1640 media. If a different glucose concentration is desired, glucose-free RPMI 1640 media may be used and a specific concentration of glucose may be added.
10. For a stock Krebs Ringer Bicarbonate Buffer (Krebs), add the following ingredients to double distilled water at the following concentrations: NaCl (118.41 mM), KCl (4.69 mM), MgSO₄ (1.18 mM), KH₂PO₄ (1.18 mM) , NaCHO₃ (25 mM), HEPES (5 mM), and CaCl₂ (2.52 mM) at pH 7.4. CaCl₂ should be added last and this solution can be stored at 4 °C for up to 2 weeks.
11. To make a working stock of Krebs, first gas with 95% O₂/182 5% CO₂ for 10-15 min with a Pasteur pipette at 37 °C followed by the addition of 0.5% RIA grade BSA by volume. Next, glucose is added to the desired concentration for the in vitro assay.

2. Preparation of Tools

1. Slightly blunt the tips of 30- and 27-G needles using a sharpening stone to round off the sharp point. Put a 45 degree bend in the needle about ¼ of an inch from the tip using a pair of pliers. Be careful not to squeeze too hard, which will close off the internal diameter of the needle.
2. Cut the 3/0 silk suture thread into approximately 4-inch lengths, one for each mouse.
3. Cover the dissecting microscope base with plastic wrap and tape down.
4. Cut several pieces of absorbent bench paper, approximately 3 x 5 inches in size, for at least 2 per mouse.

3. Preparing the Mouse

1. Fill a 1 ml syringe with 1 ml Avertin.
2. Inject the avertin solution intraperitoneally at about 40 μl/gram of body weight.
3. After the mouse no longer responds to tail or hind foot pinches, carefully transfer it to the dissecting microscope work station.
4. With the mouse in the supine position and its head facing distally, spray the mouse with 70% aqueous ethanol. Be sure to use an adequate amount of 70% ethanol to limit the amount of fur in the exposed thoracic cavity.

4. Opening the Thoracic Cavity

1. Pinch the fur and skin of the mouse between the two hind legs and make an initial cut through the epidermis, dermis and underlying membrane.
2. While still pinching the skin and underlying membrane, cut toward the front limb on both sides of the mouse taking care to avoid cutting any internal organs.
3. Fold the skin and membrane flap over the ribcage and remove the xiphoid process to allow easier access to the pancreas for inflation.
4. Reorient the mouse with the head facing proximally and move the internal organs toward the right hand side of the work station to reveal the descending aorta.
5. Unless blood tissue is to be collected, the descending aorta can be cut to complete euthanization. Soak up the excess blood with bench paper or a Kimwipe for easier access to the common bile duct.

5. Inflating the Pancreas

1. With a dissecting microscope, reposition the small intestine so the common bile duct forms a perpendicular line with the head of the mouse.
2. Take a forceps and grasp the small intestine and find the Sphincter of Oddi (Sphincter of ampulla) at the end of the common bile duct, which splays out onto the small intestine from the bile duct, forming a white triangle on the pink intestine. Once the Sphincter of Oddi has been located, take a #5 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.
3. 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 Oddi as possible, making sure the knot is taut to prevent leakage.
4. Grasp both ends of the suture and pull towards the tail to put some tension on the common bile duct. Using a free hand, make a small incision with the micro scissors in the common bile duct just above the last bifurcation into the liver. Note: It is important to not cut too close to the Sphincter of Oddi as this could result in a partial inflation of the pancreas and avoid cutting through the common bile duct as it will also result in a poor inflation.
5. While holding the two ends of string with the common bile duct taut, remove the syringe/cannula that has been pre-loaded with 5 ml collagenase solution from the ice bucket, set down the syringe, and insert the blunted 30 G needle into the cut common bile duct, being careful not to pierce the through the wall of the duct. If the 30 G needle is not wide enough for the bile duct to form a seal around, remove it and replace with the blunted 27 G needle.
6. While holding the needle in place, release the suture and pick up the 5 ml syringe. Slowly depress the plunger of the syringe. Note: If the needle has been successfully inserted into the common bile duct, it will expand.
7. Continue to depress the plunger slowly and letting off in a pulsatile fashion until the first part of the pancreas inflates, followed by gentle constant pressure until the pancreas no longer inflates. Note: Most pancreases hold 3-5 ml before starting to leak. Also, a successful inflation will have even distribution of exocrine tissue and the part of the pancreas on top of the stomach will be inflated.
8. Remove the needle from the common bile duct and set off to the side.

6. Pancreas Removal

1. Take a forceps in one hand and a curved scissors in the other. Using the forceps, grasp the small intestine at the Sphincter of Oddi. With the scissors closed, use the tip to separate part of the pancreas from the small intestine.
2. Spread the scissors apart to widen the gap between the small intestine and the pancreas.
3. Place the “V” of the scissors where the small intestine and pancreas attach to each other at the right hand side of the gap that was just made. Using the forceps gently pull the small intestine past the scissors to remove the pancreas.
4. Keep working down the small intestine to the pink connective tissue. At this point, pick up the small intestine near where it attaches to the stomach and snip the pancreas away from the rest of the small intestine. Note: Take care not to snip the pancreas as it may start to deflate.
5. Gently grab the part of the pancreas attached to the stomach with forceps (flat forceps work best). While pulling up gently on the pancreas, use the curved scissors to snip away at the connections between the pancreas and stomach.
6. Locate the spleen and hold it with the forceps above the pancreas. Take the curved scissors and cut away the connections between the spleen and the pancreas.
7. Find where the pancreas is attached to the large intestine. Pick up the large intestine with forceps and use the tip of the scissors to poke through. Spread the scissors apart as before to generate a gap between the large intestine and the pancreas.
8. Hold up the large intestine with forceps: this will result in the pancreas falling away, exposing its precise connections to the large intestine. Snip away the remaining connections.
9. Find the pink connective tissue attached to the pancreas and small intestine and cut as close to the pancreas as possible. Note: If piercing the pancreas is a concern, it is advised to cut closer to the small intestine as the connective tissue will be filtered out in later steps.
10. Grab as much of the pancreas as possible and lift it gently. With curved scissors, cut away the remaining connections between the pancreas and the thoracic cavity to fully remove the pancreas. Note: The pancreas should still be inflated if removed correctly.
11. Store the removed pancreas in ~2.5 ml of collagenase solution in a 50 ml conical tube on ice until all other pancreases have been inflated and removed for the experiment.

7. Washing

1. Take all isolated pancreases and move them to separate 50 ml conical tubes each containing 25 ml of fresh collagenase solution
2. Place the 50 ml conical tubes upright in a 37 °C shaking water bath capable of oscillating at 220-250 rpm, with the shaker turned off.
3. Gas each 50 ml conical tube with 95% O₂/5% CO₂ for 5 min with a Pasteur pipette.
4. Recap each tube tightly and place sideways in the shaking water bath underneath the surface of the water. Shake at 220-250 rpm for 20 sec, every other minute, up to 16 min starting at min 6. (Shake at 6, 8, 10, 12, 14, and 16 min)
5. Immediately transfer the tubes to a centrifuge at room temperature for a quick spin. Bring the centrifuge up to 1,500 rpm (400-500 g) and turn off immediately.
6. Using a vacuum trap, aspirate about 22.5 ml of the collagenase solution without disturbing the pellet. Wash with 10 ml HBSS and vortex gently to break up the pellet.
7. Perform another quick spin at room temperature up to 1,500 rpm (400-500 g).
8. Aspirate about 10 ml of the solution, again without disturbing the pellet.
9. Wash with another 10 ml of ice cold HBSS and vortex gently to once again break up the pellet. Repeat the quick spin and aspirate 10 ml of the solution.
10. Gently vortex the pellet in the remaining 2.5 ml of HBSS solution. Pour solution through a 1,000 μm mesh into a new 50 ml conical tube. This will remove the large tissue debris not digested by the collagenase.
11. Rinse the old 50 ml conical tube with 2.5 ml of ice cold HBSS. Use a Pasteur pipette to rinse the sides of the tube thoroughly before transferring the 2.5 ml of HBSS through the mesh into the new 50 ml conical tube.
12. Perform another quick spin at room temperature at 1,500 rpm (400-500 g) to pellet the tissue.
13. Aspirate all of the HBSS by tilting the tube toward the vacuum trap. Note: It is very important to not disturb the pellet as this will result in the loss of islets. If the pellet is loose or starts to move toward the vacuum trap, stop aspirating the solution and re-pellet the tissue and then continue to aspirate the remaining solution.
14. Once all of the HBSS has been removed, add 4.8 ml of 25% Ficoll solution and vortex to break up the pellet.
15. Carefully layer 2.4 ml of 23% Ficoll solution on top of the 25% Ficoll solution by adding the 23% Ficoll solution to the side of the 50 ml conical tube. To ensure even layering, rotate the 50 ml conical tube when adding the Ficoll solution.
16. Repeat the layering process for 20.5% and then 11% Ficoll solutions. Note: If 4 distinct layers are not present, add HBSS up to the 50 ml mark and invert the tube 4-6 times or until the Ficoll gradient no longer exists. Re-pellet the tissue and retry steps 7.14-7.16.
17. Spin the 50 ml conical tube at RT at 1,820 rpm (800 g) for 15 min.
18. Pour all of the Ficoll into a fresh 50 ml conical tube, taking care to leave the pellet of exocrine tissue behind. Add ice cold HBSS up to the 50 ml mark and invert the tube 4-6 times to mix the Ficoll and HBSS together.
19. Spin the 50 ml conical tube at RT at 1,820 rpm (800 g) for 5 min.
20. Carefully aspirate most of the HBSS/Ficoll mixture using a vacuum trap. Do not disturb the pellet as it is loose and can be aspirated easily.
21. Take a Pasteur pipette and transfer the pellet to a disposable culture tube.

8. Picking Islets

1. Add 5 ml of picking media (i.e. supplemented RPMI 1640 or Krebs Ringer Bicarbonate Buffer) to the disposable culture tube. Note: The picking media will vary depending on downstream applications.
2. Let the islets settle to the bottom of the culture tube for 5 min. Transfer them using a Pasteur pipette to a 15 mm by 60 mm Polystyrene Petri dish. Note: It is important to use a Petri dish as these are not treated and will prevent the islets from adhering to the dish.
3. In a separate 15 mm by 60 mm polystyrene Petri dish, add 5 ml of mouse islet culture media (100 ml RPMI 1640 Stock Media, 10 ml Heat Inactivated FBS, 1 ml Pen/Strep; filter sterilized).
4. With the assistance of a dissecting microscope with backlight capability, use a P-20 pipette to pick the islets into the Petri dish containing islet culture media, taking care to avoid transferring acinar tissue debris. When backlit, islets will appear brownish-gold and their outer membrane may glisten, while acinar tissue will appear light grey and dull. If the islet preparation contains a large amount of debris, it is advisable to hand-pick islets twice into fresh medium. Reducing debris contamination will limit islet clumping after an overnight incubation. Adding DNAse (3,000 U/ml) to the digestion buffer may also help to limit islet clumping (J.W.J., personal observation). Note: It is important to count the number of islets isolated to plan for downstream experiments.
5. Incubate the islets O/N in an incubator set to 37 °C with 5% CO₂.

9. cAMP Assay

1. Label 1.7 ml microfuge tubes, one for each replicate of the assay. Note: cAMP assays should have at least duplicates for each treatment condition, but more replicates per treatment are preferred.
2. After gassing the Krebs Ringer Bicarbonate Buffer for 15 min with 95% O₂/5% CO₂ with the Pasteur pipette, add 0.5% RIA grade BSA and a final glucose concentration of 1.7 mM (preincubation solution). Then, add 1 ml of the freshly gassed Krebs to each microfuge tube. Two ml of Krebs is required per tube for the pre-incubation and treatment time points; however it is recommended to prepare at least 5-10 ml extra.
3. Swirl the islets to the middle of the Petri dish, and using a P-20 pipette, hand-pick the islets to a new 15 mm by 60 mm polystyrene Petri dish containing 5 ml of the preincubation solution to wash the glucose-containing culture medium from the islets.
4. The cAMP assay kit used is the GE Healthcare cAMP Direct BioTrak EIA. The prococol used is a modification of that described for “Intracellular cAMP measurement using the non acetylation EIA procedure with the novel lysis reagents,” and has been optimized for use with the minimum number of islets per tube, allowing increased numbers of treatment conditions and technical replicates. Using a P-20 pipette, transfer at least 13 islets into each tube from step 9.2, maintaining islet size consistency between tubes, with 1 ml pre-incubation buffer. Note: Increasing the number of replicates is more beneficial than the number of islets per tube. However, if there are enough islets to increase the number of islets per tube evenly, it is advisable to do so, up to 25-50 islets per tube.
5. With the microfuge tube caps open, transfer to a 37 °C incubator set at 5% CO₂ and incubate for 45 min to normalize the insulin secretion of all islets to a baseline level.
6. While the samples are incubating, prepare the treatments (i.e. 8 mM, 11.1 mM, 16.7 mM glucose, etc.) in the gassed Krebs from step 9.2. in 15 ml conical tubes, with 1 ml extra to account for any pipetting error. Add 3-isobutyl-1-methylxanthine (IBMX) to a final concentration of 200 μM. IBMX is a general phosphodiesterase inhibitor that blocks the degradation of cAMP, allowing cAMP to accumulate in the cell as it is produced. (Note: This is a true cAMP production assay. See the discussion section for instances when measuring cAMP production may not be desirable. If IBMX is left out of the assay, more islets will be required per tube in order to obtain cAMP data in the linear range of the standard curve).
7. After the 45 min pre-incubation, remove the microfuge tubes from the incubator, cap and pulse-vortex each sample (less than 0.5 sec). This is to mix up the insulin gradient that has likely been generated due to the islets being concentrated in the bottom of the tube. Care must be taken not to vortex too harshly, as this could negatively impact the islet stability (Note: Capped tubes with islets may be gently flicked or inverted if it is not possible to pulse-vortex gently. These options will add time to the assay, which should be considered when planning the experiment).
8. Transfer each tube to a table top centrifuge (RT) and spin until the islets reach 10,000 rpm (7,000-7500 g) and then turn off immediately.
9. Use a P-1000 pipette to remove most of the Krebs in each microfuge tube, leaving approximately 10-15 μl of Krebs on the islet pellet. Use a P-20 pipette to remove the portion closest the islet pellet. If pellet is disturbed, pipette the Krebs back into the tube and re-spin.
   (Note: If the experimenter finds it too difficult to remove all of the Krebs from the islet pellet without disturbing it, the last 10-15 μl may be left on and accounted for in the total volume later in the protocol.
10. Obtain an adequate amount of liquid nitrogen in an insulated container to snap freeze the samples after incubation.
11. Take the samples out of the incubator, cap, vortex quickly (less than 0.5 sec) spin the microfuge tubes in a table top centrifuge (RT) up to 10,000 rpm (7,000-7500 g), and then shut off immediately. If insulin or another metabolite is a desired downstream application, use a P-1000 pipette to collect an aliquot of medium in a microfuge tube and store at -20 °C until further analysis. If stimulation media is not being collected, it may be discarded.
    (Note: IBMX is a strong potentiator of glucose-stimulated insulin secretion (GSIS). Therefore, GSIS results obtained from cAMP stimulation medium may not accurately represent the true effect of specific treatments on GSIS, especially if these effects are subtle).
12. Repeat step 9.9 to remove as much stimulation medium as possible without disturbing the islet pellet. Snap freeze the islet pellet in liquid nitrogen once there is less than 10-15 μl of Krebs left on the islet pellet. Once all the samples have been snap frozen, store at -80 °C until cAMP measurement.
13. On the day of cAMP determination, prepare the novel lysis reagents according to the manufacturers’ protocol. Add 200 ml lysis reagent 1B to each microfuge tube and vortex at top speed for 30 sec. Let the tubes sit on ice for 10 min, vortexing every 2 min for 30 sec. (Note: The protocol recommends carrying out a microscopic analysis with trypan blue to ensure cell lysis, but this is not performed for islets).
14. Prepare the working standards and set up the EIA microplate exactly as described in the manufacturer’s protocol. After the enzyme substrate has been incubated with the microplate for 30 min, perform the optional 1.0 M sulfuric acid step, and determine the absorbance of the microplate wells at 450 nm using a microplate reader.
15. Cyclic AMP results will be recorded as fmol/well. This should be normalized to the total volume of the original sample (200 μl + any residual Krebs left on the islets from step 9.9). Next, results can either be normalized to the total number of islets, giving fmol cAMP produced/islet, or lysate samples can be subjected to bicinchoninic acid (BCA) protein assay to normalize the results to total cellular protein (giving fmol/μg protein). The latter is recommended when islet sizes are inconsistent between samples, and can be a surrogate for islet size. The Pierce BCA protein assay kit has been used with success in this protocol, and requires only 25 ml of sample. The BSA standards should be prepared in lysis reagent 1B from the cAMP assay kit.

Wyniki

To ensure a high islet yield during isolation, surgical techniques outlined in the protocol should be followed closely. Although the techniques presented here will be tailored to each laboratory, there are a few critical steps that will lead to a successful isolation. In order to make the common bile duct easily accessible, it is recommended that the organs be displaced to the right side of the mouse (Figure 1). Moreover, this will allow the pancreas to inflate with a smaller amount of resistance since t...

Dyskusje

With the prevalence of diabetes projected to affect 7.7% of the world’s population, the requirement of novel research techniques is imperative to both understand and treat diabetes¹⁸. The present islet isolation is a well-established protocol used for in vitro experimentation and has been presented previously with slight modifications^11,14,16. Although insulin secretion is a common downstream application for isolated islets, focusing on upstream constituents, such as cAMP, may help de...

Materiały

Name	Company	Catalog Number	Comments
Collagenase from Clostridium histolyticum suitable for isolating active islets	Sigma-Aldrich	C7657
Ficoll 400	Sigma-Aldrich	F9378
Hanks Balanced Salt Solution 10X	Invitrogen (Gibco)	14065-056
HEPES	Sigma-Aldrich	H3375
RPMI 1640 (powder)	Invitrogen (Gibco)	31800-022
Albumin from Bovine Serum (BSA)	Sigma-Aldrich	A7888
3/0 Silk Suture Thread	Fine Science Tools	18020-30
Dumont #5 Forceps	Fine Science Tools	11251-10
0.8 mm Forceps	Fine Science Tools	11050-10
Curved Scissors	Fine Science Tools	14061-10
Vannas-Tübingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge	Fine Science Tools	15003-08
Dissecting Scissors	Fine Science Tools	14002-14
5 ml BD Luer-Lok Syringe	BD	309646
1 ml BD syringe	BD	309628
30 G BD Needle 1/2" Length	BD	305106
27 G BD Needle 1/2" Length	BD	305109
Sharpening Stone	Fine Science Tools	29008-01
2-2-2-tribromoethanol	Sigma-Aldrich	T48402-25G
2-methyl-2-butanol	Sigma-Aldrich	240486-100mL
Sodium Chloride (NaCl)	Sigma-Aldrich	S9888
Potassium Chloride	Sigma-Aldrich	P3911
Monopotassium Phosphate (KH2PO4)	Sigma-Aldrich	P0662
Sodium Bicarbonate (NaCHO3)	Sigma-Aldrich	S6014
CaCl2·2H2O	Sigma-Aldrich	C3881
MgSO4·7H2O	Sigma-Aldrich	M9397
Penicillin-Streptomycin	Invitrogen (Gibco)	15140-122
Heat Inactivated Fetal Bovine Serum (H.I. FBS)	Fisher Scientific	SH30088.03HI
3-Isobutyl-1-methylxanthine (IBMX)	Sigma-Aldrich	5879-100MG