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Method Article
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.
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.
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 pathology2,3. 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 vitro4,5. 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 cells4,5,6. 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 Zn2+;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 islets15,16. 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 islets14,17.
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.
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 described5,18.
1. Preparation of solutions and culture media
NOTE: Culture media, cytokine stocks, and other reagents should be prepared under sterile conditions.
2. Treatment of isolated islets with cytokines
3. Islet viability measurement with FDA/PI
4. Apoptosis measurement using staining
5. Apoptosis measurement with Annexin V/ nuclear stain
6. β-cell death measurement using Zn2+ selective indicator / nuclear stain/PI
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...
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...
The authors have no conflicts of interest to disclose.
The following grants provided funds for this work: NIDDK award R01 DK137221, JDRF 3-SRA-2023-1367-S-B to N.L.F., and ADA 7-20-JDF-020 to N.L.F. The authors would like to acknowledge support from the Diabetes Research Center at the University of Colorado Anschutz Campus P30-DK116073 and the associated core facilities utilized to support this work.
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 |
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