Isolated Pancreatic Islet Treatment and Apoptosis Measurement

Resumo

This study utilizes multi-staining fluorescence-based markers of cell death and apoptosis combined with confocal microscopy to assess cytokine-induced apoptosis and β-cell-specific death in pancreatic islets. It reveals spatial and temporal changes in cell death and apoptosis in response to extracellular stimuli.

Resumo

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing β-cells, using a combination of fluorescence staining techniques and confocal microscopy to assess cell viability, apoptosis, and β-cell-specific death. Isolated mouse islets were treated with varying concentrations of a cytokine cocktail, including TNF-α, IL-1β, and IFN-γ, to mimic immune-mediated apoptosis during the development of type 1 diabetes. The viability of islet cells was evaluated with FDA/PI dual staining, where FDA conversion to fluorescein indicated viable cells, and PI marked membrane-compromised cells. YOPRO-1 and nuclear staining provided additional data on apoptosis, with Annexin-V confirming early apoptotic cells. Quantitative analysis revealed significant increases in apoptosis and cell death rates in cytokine-treated islets. To specifically assess effects on β-cells, Zn²⁺ selective indicator staining was used to label insulin-producing cells through the zinc association in insulin granules, revealing substantial β-cell loss following treatment of islets with pro-inflammatory cytokines for 24 h. These multi-staining protocols effectively capture and quantify the extent of cytokine-induced damage in islets and can be used to evaluate therapeutics designed to prevent β-cell apoptosis in early type 1 diabetes.

Introdução

The pancreatic islets, also known as the islets of Langerhans, are a collection of endocrine cells located within the pancreas. The insulin-producing β-cells are the most abundant and functionally significant component of the pancreatic islets. These β-cells secrete insulin, a hormone that plays a critical role in maintaining glucose homeostasis¹. In type 1 diabetes (T1D), the immune system targets and infiltrates the pancreatic islets, destroying insulin-producing β-cells. This autoimmune attack is primarily mediated by pro-inflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ)². These cytokines initiate a cascade of signaling events within β-cells that ultimately trigger apoptosis³. Apoptosis, the programmed cell death, is a tightly regulated process involving the activation of caspases, DNA fragmentation, and cellular disintegration. It contributes to the gradual loss of β-cell mass and function during the onset of T1D³.

Understanding the molecular mechanisms that drive β-cell apoptosis is critical for identifying strategies to prevent or mitigate β-cell destruction in T1D. To achieve this, isolated pancreatic islets from experimental models or human cadavers serve as a robust and well-established model system for studying β-cell pathology^2,3. By treating these isolated islets with pro-inflammatory cytokines, researchers can replicate the environment that characterizes early T1D, allowing for the detailed study of β-cell dysfunction and death in vitro^4,5. These experiments provide key insights into the vulnerability and survival of β-cells under disease-associated conditions, and they also serve as a platform for testing therapeutic interventions aimed at protecting or rescuing β-cells from cytokine-induced apoptosis. By utilizing this in vitro system, we can effectively analyze how islets from different species respond to various conditions, providing a better understanding of functional and apoptotic variations across species.

Previous studies have shown that mouse and human islets treated for 24 h with a cocktail (1x = 10 ng/mL TNF-α, 5 ng/mL IL-1β, and 100 ng/mL IFN-γ) of mouse and human-derived cytokines, respectively, resulted in significant death of islet cells^4,5,6. Islet viability was confirmed by staining the cytokine-treated cells with fluorescein diacetate (FDA) and propidium iodide (PI)^5,6. Globally, islet viability is assessed using the standard deoxyribonucleic acid (DNA)-binding dye exclusion technique with FDA and PI⁷. Fluorescent stains or dye-conjugated substrates assess cell viability based on membrane integrity and permeability. FDA, a cell-permeable dye, is converted by living cells into green fluorescence (fluorescein). In contrast, PI, a cell-impermeant dye, stains only the nuclei of dead cells with compromised membranes⁷. Cells are then analyzed through two-color imaging on a confocal microscope, where green and red fluorescents mark viable and dead cells, respectively.

The limitation of the FDA/PI staining protocol is that PI only enters cells that have lost membrane selectivity, which means it cannot distinguish early apoptotic cells. Moreover, this method cannot differentiate between cell subsets, making it unsuitable for selectively assessing β-cell viability. The Annexin V/ PI protocol is commonly used for studying apoptotic cells, and the protocol has been modified to improve its accuracy⁸. The early stages of apoptosis involve the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane. Annexin V, a calcium-dependent protein, binds with high affinity to this exposed phosphatidylserine. Staining with PI is conducted alongside annexin-V to distinguish apoptotic cells (annexin V-positive only) from necrotic cells (positive for both annexin-V and PI), as necrotic cells also display phosphatidylserine due to compromised membrane integrity⁹. Other dyes, such as YOPRO-1, are also used to quantify apoptosis of islet cells. The cell membrane of viable cells is impermeable to YOPRO-1, unlike annexin-V, which cannot quantify living cells undergoing apoptosis.

To assess pancreatic β-cell death, specific dyes targeting only the insulin-producing cells are needed. A distinct feature of the pancreatic β-cells is that a portion of intracellular Zn²⁺ is stored in vesicles as a Zn²⁺-insulin complex (2:1 ratio)¹⁰. Free Zn²⁺ also exists in the extragranular space around the β-cells as reservoirs. Free Zn²⁺ and Zn²⁺ loosely bound to insulin in secretory granules can be visualized using zinc-binding dyes. Dithizone, a zinc-binding dye, is commonly used to assess islet purity, but it cannot be combined with fluorescent dyes used for evaluating β-cell viability and function¹¹. UV probes like TSQ and Zinquin that are highly selective towards Zn²⁺ have been developed to quantify β-cells by imaging and measurement of free intracellular Zn^2+;12,13. However, their use is limited by poor solubility, uneven cell loading, UV excitation requirement, and compartmentalization into acidic vesicles¹⁴. Visible wavelength fluorescent probes, such as Newport green and Zn²⁺ selective indicator, have also been developed to overcome these limitations and are now widely used to detect β-cells in isolated human islets^15,16. FluoZin-3 (Zn²⁺ selective indicator) has higher Zn²⁺ affinity and superior quantum yield than Newport Green and has proven effective for imaging Zn²⁺ co-released with insulin in isolated islets^14,17.

Using fluorescent dyes like FDA, PI, Annexin V, YOPRO-1, and Zn²⁺ selective indicator enables the measurement and differentiation between viable, apoptotic, and total dead cells. Combining compatible and highly selective probes also offers a targeted method for assessing and quantifying β-cell viability and apoptosis, which is critical for understanding and mitigating β-cell destruction in diabetes research and drug development.

Protocolo

All experiments with mice were approved by the University of Colorado Denver Institutional Animal Care and Use Committee (Protocols 000929). C57Bl/6 mice used for this experiment were purchased from the Jackson Laboratory and housed in a temperature-controlled facility on a 12 h light/dark cycle with access to food and water ad libitum. The isolated mouse islets were obtained using the collagenase digestion protocol, which has been previously described^5,18.

1. Preparation of solutions and culture media

NOTE: Culture media, cytokine stocks, and other reagents should be prepared under sterile conditions.

1. Prepare an islet culture medium by adding 10% fetal bovine serum (FBS), 10,000 U/mL penicillin, and 10,000 µg/mL streptomycin to 500 mL of 1640 RPMI Medium.
2. Prepare 1x phosphate-buffered saline solution (PBS), pH 7.4. Prepare a 1000x stock solution of mouse cytokines cocktail, 10 µg/mL TNF-α 10µg/mL, 5 µg/mL IL-1β, and 100 µg/mL IFN-γ in sterile PBS containing 0.1% bovine serum albumin (BSA) and store the solution in 10 µL aliquots at -20 °C.
3. Prepare a 46 µM (50x) FDA stock solution in acetone and store it in 1 mL aliquots at -20°C. Prepare a 1.434 mM (50x) PI stock solution in PBS and store it in 1 mL aliquots at 4 °C.
4. Prepare 500 mL of Bicarbonate-Modified Krebs-Henseleit HEPES (BMHH) buffer with 125 mM NaCl, 5.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, and 10 mM HEPES in 500 mL of dH₂O. Adjust pH to 7.4.
5. Prepare 100 mL of Annexin-V binding buffer with 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂ in 100 mL of dH₂O. Adjust the pH to 7.4.
6. Prepare 1mM Zn²⁺ selective indicator stock solution in DMSO and store in 10 µL aliquots at -20 °C.
   NOTE: Fluorescent dyes are light-sensitive, so storage and incubation must be in the dark.

2. Treatment of isolated islets with cytokines

1. Isolate mouse islets with a 10 µL micropipette into the culture medium and incubate overnight at 37 °C and 5% CO₂ to recover from isolation stress before treatment with cytokines.
2. Add 2 mL of the sterilized islet culture medium to 35 mm non-tissue culture-treated Petri dishes and label them appropriately to differentiate cytokine-treated and untreated dishes without cytokines.
3. For cytokine-treated dishes, remove 6 µL of culture medium and replace it with 2 µL of each cytokine from the stock solution to give a final relative cytokine concentration of 10 ng/mL, 5 ng/mL IL-1β, and 100 ng/mL IFN-γ (1x RCC).
   NOTE: Lower RCC of 0.5x and 0.1x can be prepared by diluting stock solutions with sterile PBS containing 0.1% BSA at 1:1 and 1:9, respectively.
4. At 12 h-24 h post-isolation, pick up 10-20 islets using a micropipette under a light microscope and transfer into the dishes and incubate in a humidified 37 °C, 5% CO₂ incubator for 24 h.
   NOTE: The incubation time may vary depending on the specific experimental objectives.

3. Islet viability measurement with FDA/PI

1. Add 20 µL each of FDA and PI stock solutions to 960 µL of the BMHH buffer containing 0.1% BSA to give a final concentration of 0.46 µM and 14.34 µM of the fluorescent dyes, respectively.
2. Aliquot 100 µL of the staining solution to a 7 mm glass-bottom non-tissue culture-treated Petri dish.
3. At 24 h post-incubation with cytokines, carefully transfer at least 6 islets from each treatment into the glass-bottom Petri dish. Incubate for 5 - 10 min at room temperature in the dark (cover with foil).
4. Take images using fluorescence microscopy with a 40x water immersion objective. Take images within 15 min of the FDA/PI staining.
5. Use excitation lasers at 488 nm and 514 nm to detect the fluorescence emissions for FDA (520 nm) and PI (620 nm), respectively.
6. Image islets as a z-stack consisting of three images, 10 µm apart. Only image the bottom ⅓ - ½ of the islet to reduce loss of signal due to light scattering issues in the islet.
7. Count live green (FDA-positive) and dead red (PI-positive) cells manually in ImageJ (NIH) for at least 5 islets per mouse (n = 3). Calculate the percentage of cell death as follows:
   percentage of islet death = number of dead cells/ (number of dead cells + number of live cells) x 100%

4. Apoptosis measurement using staining

1. Add 8 µL of YOPRO-1 (1mM solution) to 992 µL of BMHH imaging buffer for a final concentration of 0.8 µM.
2. Aliquot 500 µL of the staining solution to a 14 mm glass-bottom non-tissue culture-treated Petri dish.
3. At 24 h post-incubation with cytokines, carefully transfer at least 6 islets from each treatment into the glass-bottom Petri dish and incubate for 1 h at 37 °C in the dark (cover with foil).
4. After 20 min of incubation, add a drop of NucBlue (nuclear stain, 20 µL) to each glass-bottom Petri dish and continue incubation. Total incubation time is 1 h for YOPRO-1 and 40 min for nuclear stain.
5. After 1 h of incubation, transfer islets to a fresh BMHH imaging buffer containing 0.1% BSA (100 µL) in a 7 mm glass-bottom non-tissue culture-treated Petri dish.
6. Take images using fluorescence microscopy with a 40x water immersion objective. Use excitation lasers at 405 nm and 488 nm to detect the fluorescence emissions of nuclear stain (460 nm) and YOPRO-1 (509 nm), respectively.
7. Image islets as a z-stack consisting of three images, 10 µm apart. Only image the bottom ⅓ - ½ of the islet to reduce loss of signal due to light scattering issues in the islet.
8. Count live blue (nuclear stain-positive) and apoptotic green (YOPRO-1-positive) cells manually in ImageJ (NIH) for at least 5 islets per mouse (n = 3). Calculate the percentage of apoptotic islet cells as follows:
   percentage of apoptotic islet cells = number of apoptotic cells / (number of apoptotic cells + number of live cells) x 100%

5. Apoptosis measurement with Annexin V/ nuclear stain

1. Add 2 drops of nuclear stain (40 µL) to 1 mL of Annexin binding buffer. Aliquot 100 µL of the solution into a 7 mm-glass-bottom non-tissue culture-treated Petri dish.
2. At 24 h post-incubation with cytokines, carefully rinse by pipetting up and down 3x at least 6 islets from each treatment in PBS by pipetting up and down thrice using the 10 µl micropipette. Transfer the islets to the glass-bottom Petri dish with the nuclear stain solution and incubate them for 40 min at 37 °C in the dark (cover with foil).
3. After 25 min of incubation, add 5 µL of Annexin V Alexa Flour 488 conjugate to each glass-bottom petri dish and continue incubation. Total incubation time is 40 min for the nuclear stain and 15 min for Annexin V.
4. After 40 min of incubation, transfer islets to a fresh BMHH imaging buffer (without Annexin V or nuclear stain) in a 7 mm glass-bottom Petri dish.
5. Take images using fluorescence microscopy with a 40x water immersion objective. Use excitation lasers at 405 nm and 488 nm to detect the fluorescence emissions of nuclear stain (460 nm) and Annexin V conjugate (515 nm), respectively.
6. Image islets as a z-stack consisting of three images, 10 µm apart. Only image the bottom ⅓ - ½ of the islet to reduce loss of signal due to light scattering issues in the islet.
7. Count live blue (nuclear stain-positive) and apoptotic green (Annexin V-positive) cells manually in ImageJ (NIH) for at least 5 islets per mouse (n = 3). Calculate the percentage of apoptotic islet cells as follows:
   percentage of apoptotic cells = number of apoptotic cells / (number of apoptotic cells + number of live cells) x 100%

6. β-cell death measurement using Zn²⁺ selective indicator / nuclear stain/PI

1. Add 2 µL of Zn²⁺ selective indicatorAM stock solution to 998 µL of BMHH imaging buffer to make a final concentration of 0.2 µM. Aliquot 500 µL of the staining solution to a 14 mm glass-bottom non-tissue culture-treated Petri dish.
2. At 24 h post-incubation with cytokines, carefully transfer at least 6 islets from each treatment into the glass-bottom Petri dish. Incubate for 1 h at 37 °C in the dark with the Zn²⁺ selective indicator AM solution (cover with foil).
3. After 20 min of incubation, add a drop of nuclear stain (20 µl) to each glass-bottom Petri dish, per manufacturer's instructions and continue incubation. Total incubation time is 1 h for Zn²⁺ selective indicator and 40 min for nuclear stain.
4. After 1 h of incubation, transfer islets to a fresh BMHH imaging buffer containing 0.1% BSA (100 µL) in a 7 mm glass-bottom non-tissue culture-treated Petri dish. Add 2 µL of PI stock solution for 10 min to make a final concentration of 20 µg/mL.
5. Take images within 15 min of the PI staining using fluorescence microscopy with a 40x water immersion objective. Use excitation lasers at 405 nm, 488 nm, and 514 nm to detect the fluorescence emissions of nuclear stain (460 nm), Zn²⁺ selective indicator (516 nm), and PI (620 nm), respectively.
6. Image islets as a z-stack consisting of three images, 10 µm apart. Only image the bottom ⅓ - ½ of the islet to reduce loss of signal due to light scattering issues in the islet.
7. Count live blue (nuclear stain-positive), Zinc green (Zn²⁺ selective indicator-positive), and dead red (PI-positive) cells manually in ImageJ (NIH) for at least 5 islets per mouse (n = 3). Calculate the percentage of β-cell death as follows:
   percentage of β-cell death = number of zinc-positive and PI-positive cells / (number of dead + live islet cells) x 100%
   or
   percentage of β-cell death = number of zinc-positive and PI-positive cells / (number of zinc-positive cells) x 100%

Resultados

The dual staining with FDA and PI was used to assess the viability of islets treated with cytokines. All experiments were conducted in triplicate (n = 3), and data was generated from multiple z-stack images of 10 µm apart, with each replicate containing average data from 5 or 6 islets, to ensure reproducibility and statistical comparison among the treated and untreated islets. Figure 1A shows healthy islets from the untreated control group exhibiting distinct green fluorescence due to t...

Discussão

This study demonstrates the effectiveness of multi-staining methods with florescent dyes and confocal microscopy in assessing islet cell viability, apoptosis, and β-cell survival under cytokine-induced stress. FDA/PI staining revealed a dose-dependent increase in cell death within islets exposed to cytokines, as evident by red fluorescence marking membrane-compromised cells, confirming the cytotoxic effect of cytokines on cell viability. FDA/PI staining also identifies viable and dead cells in human islets, offering...

Materiais

Name	Company	Catalog Number	Comments
14 mm glass-bottom Petri dish	Mattek	P35G-1.5-14-C
1640 RPMI	Corning	10-041-CV
35 mm Petri dish	Celltreat	229638
7 mm glass-bottom Petri dish	Mattek	P35G-1.5-7-C
Annexin V, Alexa Flour 488	Thermo Fisher (Invitrogen)	A13201	ex488/em515
Calcium Chloride Dihydrate	Fisher	C79
Dimethyl Sulfoxide Anhydrous	Sigma	276855
Fetal Bovine Serum	Thermo Fisher (Gibco)	26140079
Flouzin-3, AM	Thermo Fisher (Invitrogen)	F24195	ex488/em516
Fluorescein Diacetate (FDA)	Thermo Fisher (Invitrogen)	F1303	ex488/em520
HEPES	Sigma	54457
Image processing software	NIH	ImageJ
Magnesium Chloride	Sigma	M8266
NucBlue Live ReadyProbes Reagent (Hoechst 33342)	Thermo Fisher (Invitrogen)	R37605	ex360/em460
Penicillin-Streptomycin	Thermo Fisher (Gibco)	15-140-122
Phosphate-buffered Saline Tablets	Fisher	BP2944-100
Potassium Chloride, Granular	Macron	6858-04
Propidium iodide	Thermo Fisher (Invitrogen)	P1304MP	ex535/em620
Protein Recombinant Mouse IFN-gamma Protein	R&D Systems	485-MI-100/CF
Recombinant Mouse IL-1 beta/IL-1F2	R&D Systems	401-ML-100/CF
Recombinant Mouse TNF-alpha (aa 80-235) Protein	R&D Systems	410-MT-100/CF
Sodium Chloride, Crystal	Macron	7581-12
Stellaris Confocal microcope with spectral detectors	Leica	DMI-8	40x water immersion objective.
Yopro-1 Iodide	Thermo Fisher (Invitrogen)	Y3603	ex488/em509