The overall goal of this assay is to visualize insulin granule exocytosis in real time in intact pancreatic islets by confocal microscopy. This method will answer key questions in the diabetes field. For example, what regulates insulin exocytosis?
This is critical because granule fusion is the single most important event in the life of a better cell. The main advantage of this technique is that we can use intact islets, we can see fusion events in cells within their native environment. Madina Mahkmutova, a PhD student from our laboratory will be demonstrating the technique.
The begin, upon arrival of pancreatic islets, transfer them to 50 milliliter falcon tubes and centrifuge the islets suspension at 1, 000 rotations per minute for five minutes. The islets will collect at the bottom of the tube, transfer the islets in two milliliters of CMRL culture medium to 35 millimeter non-tissue culture treated petri dishes and incubate them at 37 degree celsius and 5%carbon dioxide for 24 hours before viral infection. Following their isolation, re-suspend approximately mouse pancreatic islet equivalents in two milliliters of CMRL culture medium and transfer them to 35 millimeter non-tissue culture petri dishes.
Incubate the tissue at 37 degree celsius and 5%carbon dioxide for 24 hours before viral infection. To carry out viral infection, add five to 10 microliters of stock virus to each 35 millimeter petri dish containing human or mouse islets in two milliliters of CMRL culture medium. After culturing the islets for 24 hours, aspirate the medium and replace it with two milliliters of CMRL culture medium without virus.
Culture the islets for four to six days, replacing the medium every three days. Prepare extracellular solution by combining the reagents listed here and then filter sterilize the buffer. Make basal glucose medium to a three millimolar final glucose concentration, by adding 75 microliters of two molar glucose stock to 50 milliliters of extracellular solution.
Then to prepare hyperglycemic medium to a 16 millimolar final glucose concentration, add 400 microliters of two molar glucose stock to 50 milliliters of extracellular solution. Dilute any additional stimuli and extracellular solution containing three millimolar glucose. Before starting an experiment, pre treat cover slips with 30 microliters of one milligram per milliliter poly-d-lysine solution and incubate for one hour.
Then use water to thoroughly rinse the cover slips. At least one hour before the experiment, transfer a group of islets to a 35 millimeter petri dish containing extracellular solution with three millimolar glucose. Keep the remaining islets at 37 degrees celsius and 5%carbon dioxide.
30 minutes before starting an experiment, attach the cover slip to the imaging chamber by using silicon grease to seal it. Then fix the imaging chamber to the imaging platform. Next, using a pipette, transfer 20 to 30 islets to the poly-d-lysine treated area of the cover slip and let the islets adhere to the surface for 20 minutes.
This step is critical for the success of the experiment and we collect approximately 20 to 30 islets in less than 10 microliters of buffer and wait for 20 minutes. It is important to not let the cover slip dry completely, to avoid islet damage. While the islets are adhering to the cover slip, prepare the perfusion system by using water to thoroughly rinse it.
Then add each solution to a different channel, according to this table. Remove all the bubbles was from the system by opening each channel separately and letting the solution flow for a few minutes. Ensure that the flow is consistent and the tubing is not leaking.
Next, connect to the single inline solution heater to the perfusion outlet tube and adjust the temperature of the out flowing buffer to 37 degree celsius. Then, prepare the suction pump. Connect the tubing, turn on the pump and make sure the liquid is being aspirated.
Place the imaging platform with the islets onto the microscope stage and connect it to the perfusion system and suction pump. Then, gently fill up the imaging chamber with the extracellular solution containing three millimolar glucose. To carry out confocal imaging, locate the islets in the microscope field with low magnification.
Once focused on the islets, switch to higher magnification objectives. Open the acquisition software and activate the resident scanning mode. Select xyzt imaging, then to configure the acquisition settings, turn on the argon laser and set it to 30%and then the 488 nanometer laser line, adjusting the laser power to around 50%for pHluorin excitation.
Collect emission at 505 to 555 nanometers, using the EGFP emission spectrum as guide. Choose a resolution of 512 by 512 pixels. Start imaging by pressing the live button and adjust the gain levels.
Set the beginning end of the z stack by focusing on the top of islet and choosing begin and then moving to the last plane that can be focused and choosing n. Choose a z stack size of five micrometers. Set the time interval for acquisition of each z stack close to 1.5 to 2 seconds and choose the option, acquire until stopped for continuous imaging.
Then, press the start button to initialize imaging. Refer to the text protocol for stimulation protocols to induce granule exocytosis. Using the resident scanner, confocal images of the islet are acquired as timelapse recordings in less than two seconds.
In addition, this technique can be combined with dyes for functional imaging, like membra potential or cytosolic free calcium dyes. To determine if NP Y-pHluorin is indeed a suitable tool to monitor insulin granule dynamics, infected islets were immunostained with antibodies against GFP for NP Y-pHluorin and the islet hormones insulin, somatostatin or glucagon. Most cells expressing NP Y-pHluorin were beta cells as they also expressed insulin.
Only a few glucagon positive alpha cells or somatostatin positive delta cells were GFP labeled. Importantly in infected cells, the NP Y fusion co-localized with insulin. This figure demonstrates that pHluorin is an effective pH sensor.
As increasing the intercellular pH with 50 millimolar ammonium chloride resulted in a greater than 500%increase in the intercellular fluorescence. As the pH inside the granule increases upon fusion with the plasma membrane in the opening of the fusion pore, granules containing NP Y-pHluorin become visible under conditions that stimulate insulin exocytosis such as, membrane de-polarization with potassium chloride. Using confocal timelapse imaging of NP Y-pHluorin infected islets, single secretarial events in beta cells within intact islets can be visualized.
These occur in response to direct membrane de-polarization with potassium chloride or several other physiological stimuli. At the basal extracellular glucose concentration, little secretarial activity is observed. However, granule fusion with the plasma membrane is triggered in response to stimulation with high glucose.
Following this procedure, other methods like immunohistochemistry can be performed to answer additional questions like, why granule fusion occurs in specific regions of the cell. After its development, this technique has been used to explore the temporal pattern of insulin granule secretion in beta cell populations, in human islets.
