Isolated Pancreatic Islet Treatment and Apoptosis Measurement

My research is focused around understanding the factors that lead to development and progression of type one diabetes, and engineering new therapies to provide treatment options for patients with T1D. Our lab uses designer biomaterials as a tool to study type one diabetes in human tissues, and to develop customized drug delivery therapies for each stage of T1D. Our protocol provides detailed information about the mechanisms and spatial localization of cell death in intact islets that helps us to better understand the causes of islet death in T1D.

[Narrator] To begin, obtain mouse islets, and using a 10 microliter micropipette, isolate them into the culture medium. Incubate overnight at 37 degrees Celsius and 5% carbon dioxide to allow recovery from isolation stress before cytokine treatment. Add two milliliters of sterilized islet culture medium to 35 millimeter non-tissue, culture-treated Petri dishes. Label the dishes appropriately to differentiate cytokine-treated from untreated dishes. For cytokine-treated dishes, remove six microliters of culture medium. Replace with two microliters of each cytokine from the stock solution to achieve the desired final concentration. At 12 to 24 hours post-isolation, pick up 10 to 20 islets using a micropipette under a light microscope. Transfer the islets to the prepared dishes and incubate in a humidified 37 degrees Celsius 5% carbon dioxide incubator for 24 hours. To perform islet viability measurement using FDA and PI, add 20 microliters of FDA and PI stock solutions each to 960 microliters of BMHH buffer containing 0.1% bovine serum albumin. Aliquot 100 microliters of the staining solution into a seven millimeter glass bottom non-tissue, culture-treated Petri dish. At 24 hours post-incubation with cytaokines, carefully transfer at least six islets from each treatment into the glass bottom Petri dish. Incubate the islets for five to 10 minutes at room temperature in the dark by covering the dish with foil. Capture images using fluorescence microscopy with a 40x water immersion objective. Acquire images within 15 minutes of fluorescein diacetate and propidium iodide staining. For apoptosis measurement using staining, first add eight microliters of YOPRO-1 from a one millimolar stock solution to 992 microliters of BMHH imaging buffer. Aliquot five 500 microliters of the staining solution into a 14 millimeter glass bottom, non-tissue, culture-treated Petri dish. At 24 hours post-incubation with cytokines, carefully transfer at least six islets from each treatment into the glass bottom Petri dish. Incubate the islets for one hour at 37 degrees Celsius in the dark by covering the dish with foil. After 20 minutes of incubation, add 20 microliters of NucBlue nuclear stain to each glass bottom Petri dish. Continue incubation to reach a total incubation time of one hour for YOPRO-1, and 40 minutes for the nuclear stain. Then, transfer the islets to a seven millimeter glass bottom, non-tissue, culture-treated Petri with 100 microliters of fresh BMHH imaging buffer containing 0.1% BSA. Capture images using fluorescence microscopy with a 40x water immersion objective. Use excitation lasers at specified wavelengths to detect fluorescence emissions of the dyes. Add two drops or 40 microliters of nuclear stain to one milliliter of Annexin binding buffer. Aliquot 100 microliters of the solution into a seven millimeter glass bottom, non-tissue, culture-treated Petri dish. After 24 hours post-incubation with cytokines, using a 10 microliter micropipette, carefully rinse at least six islets from each treatment in PBS by pipetting up and down thrice. Transfer the islets to the glass bottom Petri dish containing the nuclear stain solution, and incubate for 40 minutes at 37 degrees Celsius in the dark by covering the dish with foil. After 25 minutes of incubation, add five microliters of Annexin V Alexa Fluor 488 conjugate to each glass bottom Petri dish. Continue incubation to reach a total incubation time of 40 minutes for the nuclear stain and 15 minutes for Annexin V. After 40 minutes of incubation, transfer the islets to a fresh BMHH imaging buffer without Annexin V or nuclear stain in a seven millimeter glass bottom Petri dish. Capture images with fluorescence microscopy using a 40x water immersion objective and excitation lasers at specified wavelengths. Add two microliters of zinc ion selective indicator AM stock solution to 998 microliters of BMHH imaging buffer to a final concentration of 0.2 micromolar. Aliquot 500 microliters of the staining solution into a 14 millimeter glass bottom, non-tissue, culture-treated Petri dish. At 24 hours post-incubation with cytokines, carefully transfer at least six islets from each treatment into the glass bottom Petri dish. Incubate for one hour at 37 degrees Celsius in the dark by covering the dish with foil. After 20 minutes of incubation, add 20 microliters of nuclear stain to each Petri dish and continue incubation as mentioned. Then, transfer the islets to a seven millimeter glass bottom, non-tissue, culture-treated Petri with 100 microliters of fresh BMHH imaging buffer containing 0.1% BSA. Add two microliters of PI stock solution to achieve a final concentration of 20 micrograms per milliliter and incubate for 10 minutes. Capture images within 15 minutes of propidium iodide staining using fluorescence microscopy with a 40x water immersion objective and excitation lasers at specified wavelengths. After dual staining with FDA and PI, healthy islets from the untreated control group exhibited strong green fluorescence, indicating high viability with intact cell membranes. Cytokine-treated islets displayed prominent red fluorescence, signifying increased cell death due to compromised membrane integrity. A dose-dependent increase in islet cell death was observed with rising cytokine concentrations, with higher exposure leading to increased red fluorescence. After YOPRO-1 and nuclear staining, the untreated islets showed strong blue nuclear fluorescence without green staining, confirming the presence of viable non-apoptotic cells. Cytokine treated islets displayed green fluorescence from YOPRO-1 alongside blue nuclear staining, indicating apoptotic cells. Quantitative analysis showed that more than 30% of pancreatic islets underwent apoptosis within 24 hours of cytokine exposure. Annexin V staining also confirmed apoptosis, with approximately 40% of islets showing positive staining for early and progressing apoptotic cells. The zinc selective indicator showed discrete punctate staining in untreated islets, indicating binding to zinc associated with insulin in secretory granules. Cytokine-treated islets exhibited additional red and blue fluorescence, allowing precise visualization of beta cell death. Quantitative data revealed that cytokines destroyed approximately 63% of insulin-producing beta cells within 24 hours of exposure.