Isolated Pancreatic Islet Treatment and Apoptosis Measurement

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 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.

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%

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 the conversion of FDA to fluorescein by enzymatically active cells. The absence or minimal presence of red fluorescence confirms the viability of these islets, indicating intact cell membranes. In contrast, Figure 1B demonstrates significant red fluorescence in cytokine-treated islets, marking dead or dying cells with compromised membrane integrity. This increase in red fluorescence highlights the PI staining of nuclei in non-viable cells with membrane disruption. The impact of cytokine concentration on islet viability is shown in Figure 1C. The quantitative data reveals a positive correlation between the islet death rate and increasing cytokine concentrations (0.1x-1x RCC). This dose-dependent response underscores the cytotoxic effects of cytokines on islet cells, as evidenced by the increased red fluorescence in PI-stained cells with higher cytokine exposure.

To measure cytokine-induced apoptosis, untreated and cytokine-treated islet cells were stained with YOPRO-1 and nuclear stain to quantify apoptotic and viable cells. Figure 2A shows a representative image of a non-apoptotic cell from the untreated control dish, where strong blue fluorescence is observed in the nucleus, with no green fluorescence, indicating a viable, non-apoptotic cell. Figure 2B presents cytokine-treated islets with distinct green fluorescence from YOPRO-1 alongside blue nuclear staining. This dual fluorescence confirms apoptosis, as YOPRO-1 permeates stressed cell membranes. The quantification revealed that more than 30% of pancreatic islets displayed apoptosis within 24 h of cytokine exposure (Figure 2C). Annexin-V staining was performed to validate apoptosis further, showing that approximately 40% of islets stained positively for Annexin-V, capturing both early apoptotic cells and those progressing through apoptosis (Figure 2D-F).

Finally, to assess the pancreatic β-cell death rate in response to cytokines, we stained the untreated and cytokine-treated islets with Zn²⁺ selective indicator, nuclear stain, and PI (Figure 3A,B). Our results showed a discrete punctate staining pattern with the Zn²⁺ selective indicator, indicating that the Zn²⁺ selective indicator is bound to free and loosely bound zinc associated with insulin in secretory granules. In addition, triple staining with these dyes allows for the combination of distinct fluorescence channels (blue for nuclear stain, red for PI, and green for Zn²⁺ selective indicator), enabling precise, multiplexed visualization under a fluorescence microscope, making it easy to distinguish not only live from dead cells but also specific death of the insulin-producing β-cells. It also provides data for the descriptive quantification of β-cell death as a fraction of the total number of β-cells, dead cells, or islets cells. Figure 3C shows that cytokines cause more destruction of the β-cells, destroying about 63% of the insulin-producing cells within 24 h of co-culture with healthy islets.

Figure 1: Dual staining of islets with fluorescein diacetate (green) and propidium iodide (red) to assess cytokine-mediated death. (A) Representative fluorescence microscopy images of untreated mouse islets and (B) cytokine-treated islets stained with fluorescein diacetate (green) and propidium iodide (red). (C) Percentage viability of mouse islets treated with varying relative cytokine concentrations (0.1x red squares, 0.5x green triangles, and 1x RCC blue triangles) and untreated islets (black circles). 1x RCC = 10 ng/mL TNF-α, 5 ng/mL IL-1β, and 100 ng/mL IFN-γ. Scale bar = 10 µm. Each data point is an islet. Data is presented as a mean ± SEM. *p < 0.05 and ***p < 0.001 indicate statistical significance based on ANOVA with Tukey's post-hoc analysis (n = 3). Please click here to view a larger version of this figure.

Figure 2: Staining of islets with YOPRO-1 and Annexin-V as markers of cytokine-induced apoptosis. (A) Representative fluorescence microscopy images of untreated mouse islets and (B) cytokine-treated islets stained with nuclear stain (blue) and YOPRO-1 (green). Scale bar = 10 µm. (C) Percentage of YOPRO-1 positive cells in cytokine-treated islets (1x RCC blue triangles) and untreated islets (black circles). (D) Representative fluorescence microscopy images of untreated mouse islets and (E) cytokine-treated islets stained with nuclear stain (blue) and Annexin V (green). (F) Percentage of Annexin-V positive cells in cytokine-treated islets (1x RCC blue triangles) and untreated islets (black circles). 1x RCC = 10 ng/mL TNF-α, 5 ng/mL IL-1β, and 100 ng/mL IFN-γ. Scale bar = 10 µm. Each data point is an islet. Data is presented as a mean ± SEM. ****p<0.0001 indicates statistical significance based on independent Welch's t-test (n = 3). Please click here to view a larger version of this figure.

Figure 3: Triple staining of islets with nuclear stain (blue), Zn²⁺ selective indicator(green), and propidium iodide (Red) for evaluation of specific pancreatic β-cell death. (A) Representative fluorescence microscopy images of (A) untreated mouse islets and (B) cytokine-treated islets at different z-stacks of 10 µm apart. (C) Percentage death of β-cell in cytokine-treated islets (1x RCC). 1x RCC = 10 ng/mL TNF-α, 5 ng/mL IL-1β, and 100 ng/mL IFN-γ. Scale bar = 10 µm. PI+ = Total dead cells, PI+β+ = Dead β-cells and β+ = Total (dead and live) β-cells. Each data point is an islet. Data is presented as a mean ± SEM. ****p<0.0001 indicates statistical significance based on independent Welch's t-test (n = 3). Please click here to view a larger version of this figure.

Table 1: List of fluorescent probes used in this protocol, their excitation/emission, and the cell state indicated by positive staining.

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 a quick qualitative assessment of human islet preparation viability⁷. Thus, this staining method is reliable for assessing cell viability in inflammatory conditions. This result also reinforces the role of cytokines in the pathophysiology of diabetes, where prolonged cytokine exposure is detrimental to β-cell function and survival^5,19.

The YOPRO-1/ nuclear stains and Annexin-V/ nuclear stains provided additional insights into cytokine-induced apoptosis. Cytokine-treated islets displayed increased green fluorescence indicative of dead cells associated with apoptotic cells. Annexin-V also confirmed the presence of early and progressing apoptotic cells. Together, these staining techniques effectively characterize apoptosis in β-cells, validating apoptosis as a primary mechanism of islet cell death when exposed to pro-inflammatory cytokines³.

Protein kinase inhibitors that could disrupt apoptotic signal transduction pathways, as well as antibodies and antagonists against the actions of pro-inflammatory cytokines, are being explored as promising therapeutics that could prevent β-cell apoptosis^5,20,21. These research activities employ fluorescence-based microscopy to evaluate potential interventions to mitigate cytokine-induced apoptosis, focusing on preserving β-cell function in diabetes models. By further incorporating Zn²⁺ selective indicator staining, this approach enables selective visualization of insulin-producing β-cells through zinc association, providing a targeted view of cytokine effects on β-cell populations within islets and enhancing the specificity of the analysis¹⁶. This approach distinguished β-cells from other islet cells and revealed significant β-cell destruction, with approximately 63% of β-cells death after cytokine exposure in our study. This specificity is critical for evaluating β-cell preservation and further highlights the need for its protective strategies in diabetes management.

These results underscore the versatility of FDA/PI, YOPRO-1/ nuclear stain, and Zn²⁺ selective indicator/ nuclear stain/PI staining in evaluating islet death. One critical step in this protocol is selection of the appropriate combination of fluorescent probes to minimize spectral overlap in data collection. Table 1 outlines all of the fluorescent probes demonstrated in this protocol, along with their respective excitation and emission maxima and the cell state (live/dead/apoptotic) that each stain indicates. Optimization of excitation and emission filters may be required to minimize spectral overlap between dyes to accurately quantify live, dead, or apoptotic cells. Additionally, the viability of isolated islets decreases rapidly over time, for best results in analyzing the effects of compounds on islet viability, experiments should be performed within 72 h after isolation. By providing precise, reproducible data on cell viability, apoptosis, and β-cell-specific death, this study illustrates the potential of these techniques in developing therapeutic interventions aimed at reducing cytokine toxicity and preserving β-cell function in diabetes. Published studies from our laboratory have used this approach to provide quantitative data on β-cell viability and protection against apoptosis in the presence of a small molecule inhibitor^5,21.

One limitation of this protocol is that staining of beta cell-apoptotic death can only be achieved using additional beta-cell-specific markers that do not spectrally overlap with YOPRO-1 or Annexin-V fluorescence dyes. Alternatively, more robust though expensive reporter mouse lines, such as the insulin-cre reporter mice with apoptosis-specific promoters, can also be adopted. This knockout model combines an insulin promoter-driven Cre-recombinase with a reporter system like tdTomato and GFP to visualize apoptosis in insulin-expressing pancreatic beta cells. Another limitation is that manual counting of islet images is time-consuming and has low throughput. While efforts are underway in our lab to develop a suitable ImageJ plugin to provide automated live/dead cell quantification, currently available cell counting plugins using ImageJ (NIH) or other commercially available cell-counting software work best with dyes such as nuclear stain and PI, which identify cell nuclei and have distinguishable boundaries between individual cells. Dyes like FDA and AnnexinV have more sparse straining, making it difficult to identify boundaries between cells when counting with automated software.

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.