Name: Isolated Pancreatic Islet Treatment and Apoptosis Measurement (Video) | JoVE Journal
Uploaded: 2025-05-02T13:50:00.000+00:00
Duration: 9 min 36 s
Description: Learn multi-staining fluorescence techniques to study apoptosis in mouse pancreatic islets under cytokine treatment. This method reveals spatial patterns of β-cell death relevant to type 1 diabetes research.

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5 minutes

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.

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.
<ol>
	<li>Prepare an islet culture medium by adding 10% fetal bovine serum (FBS), 10,000 U/mL penicillin, and 10,000 &#181;g/mL streptomycin to 500 mL of 1640 RPMI Medium.</li>
	<li>Prepare 1x phosphate-buffered saline solution (PBS), pH 7.4. Prepare a 1000x stock solution of mouse cytokines cocktail, 10 &#181;g/mL TNF-&#945; 10&#181;g/mL, 5 &#181;g/mL IL-1&#946;, and 100 &#181;g/mL IFN-&#947; in sterile PBS containing 0.1% bovine serum albumin (BSA) and store the solution in 10 &#181;L aliquots at -20 &#176;C.</li>
	<li>Prepare a 46 &#181;M (50x) FDA stock solution in acetone and store it in 1 mL aliquots at -20&#176;C. Prepare a 1.434 mM (50x) PI stock solution in PBS and store it in 1 mL aliquots at 4 &#176;C.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare 1mM Zn2+ selective indicator stock solution in DMSO and store in 10 &#181;L aliquots at -20 &#176;C. 
	NOTE: Fluorescent dyes are light-sensitive, so storage and incubation must be in the dark.</li>
</ol>
2. Treatment of isolated islets with cytokines
<ol>
	<li>Isolate mouse islets with a 10 &#181;L micropipette into the culture medium and incubate overnight at 37 &#176;C and 5% CO2 to recover from isolation stress before treatment with cytokines.</li>
	<li>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.</li>
	<li>For cytokine-treated dishes, remove 6 &#181;L of culture medium and replace it with 2 &#181;L of each cytokine from the stock solution to give a final relative cytokine concentration of 10 ng/mL TNF-&#945;, 5 ng/mL IL-1&#946;, and 100 ng/mL IFN-&#947; (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.</li>
	<li>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 &#176;C, 5% CO2 incubator for 24 h. 
	NOTE: The incubation time may vary depending on the specific experimental objectives.</li>
</ol>
3. Islet viability measurement with FDA/PI
<ol>
	<li>Add 20 &#181;L each of FDA and PI stock solutions to 960 &#181;L of the BMHH buffer containing 0.1% BSA to give a final concentration of 0.46 &#181;M and 14.34 &#181;M of the fluorescent dyes, respectively.</li>
	<li>Aliquot 100 &#181;L of the staining solution to a 7 mm glass-bottom non-tissue culture-treated Petri dish.</li>
	<li>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).</li>
	<li>Take images using fluorescence microscopy with a 40x water immersion objective. Take images within 15 min of the FDA/PI staining.</li>
	<li>Use excitation lasers at 488 nm and 514 nm to detect the fluorescence emissions for FDA (520 nm) and PI (620 nm), respectively.</li>
	<li>Image islets as a z-stack consisting of three images, 10 &#181;m apart. Only image the bottom &#8531; - &#189; of the islet to reduce loss of signal due to light scattering issues in the islet.</li>
	<li>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%</li>
</ol>
4. Apoptosis measurement using staining
<ol>
	<li>Add 8 &#181;L of YOPRO-1 (1mM solution) to 992 &#181;L of BMHH imaging buffer for a final concentration of 0.8 &#181;M.</li>
	<li>Aliquot 500 &#181;L of the staining solution to a 14 mm glass-bottom non-tissue culture-treated Petri dish.</li>
	<li>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 &#176;C in the dark (cover with foil).</li>
	<li>After 20 min of incubation, add a drop of NucBlue (nuclear stain, 20 &#181;L) to each glass-bottom Petri dish and continue incubation. Total incubation time is 1 h for YOPRO-1 and 40 min for nuclear stain.</li>
	<li>After 1 h of incubation, transfer islets to a fresh BMHH imaging buffer containing 0.1% BSA (100 &#181;L) in a 7 mm glass-bottom non-tissue culture-treated Petri dish.</li>
	<li>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.</li>
	<li>Image islets as a z-stack consisting of three images, 10 &#181;m apart. Only image the bottom &#8531; - &#189; of the islet to reduce loss of signal due to light scattering issues in the islet.</li>
	<li>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%</li>
</ol>
5. Apoptosis measurement with Annexin V/ nuclear stain
<ol>
	<li>Add 2 drops of nuclear stain (40 &#181;L) to 1 mL of Annexin binding buffer. Aliquot 100 &#181;L of the solution into a 7 mm-glass-bottom non-tissue culture-treated Petri dish.</li>
	<li>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 &#181;l micropipette. Transfer the islets to the glass-bottom Petri dish with the nuclear stain solution and incubate them for 40 min at 37 &#176;C in the dark (cover with foil).</li>
	<li>After 25 min of incubation, add 5 &#181;L 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Image islets as a z-stack consisting of three images, 10 &#181;m apart. Only image the bottom &#8531; - &#189; of the islet to reduce loss of signal due to light scattering issues in the islet.</li>
	<li>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%</li>
</ol>
6. &#946;-cell death measurement using Zn2+ selective indicator / nuclear stain/PI
<ol>
	<li>Add 2 &#181;L of Zn2+ selective indicatorAM stock solution to 998 &#181;L of BMHH imaging buffer to make a final concentration of 0.2 &#181;M. Aliquot 500 &#181;L of the staining solution to a 14 mm glass-bottom non-tissue culture-treated Petri dish.</li>
	<li>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 &#176;C in the dark with the Zn2+ selective indicator AM solution (cover with foil).</li>
	<li>After 20 min of incubation, add a drop of nuclear stain (20 &#181;l) to each glass-bottom Petri dish, per manufacturer&#39;s instructions and continue incubation. Total incubation time is 1 h for Zn2+ selective indicator and 40 min for nuclear stain.</li>
	<li>After 1 h of incubation, transfer islets to a fresh BMHH imaging buffer containing 0.1% BSA (100 &#181;L) in a 7 mm glass-bottom non-tissue culture-treated Petri dish. Add 2 &#181;L of PI stock solution for 10 min to make a final concentration of 20 &#181;g/mL.</li>
	<li>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.</li>
	<li>Image islets as a z-stack consisting of three images, 10 &#181;m apart. Only image the bottom &#8531; - &#189; of the islet to reduce loss of signal due to light scattering issues in the islet.</li>
	<li>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 &#946;-cell death as follows: 
	percentage of &#946;-cell death = number of zinc-positive and PI-positive cells / (number of dead + live islet cells) x 100% 
	or 
	percentage of &#946;-cell death = number of zinc-positive and PI-positive cells / (number of zinc-positive cells) x 100%</li>
</ol>

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The authors have no conflicts of interest to disclose.

This study demonstrates the effectiveness of multi-staining methods with florescent dyes and confocal microscopy in assessing islet cell viability, apoptosis, and &#946;-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 pancreatic islets, also known as the islets of Langerhans, are a collection of endocrine cells located within the pancreas. The insulin-producing &#946;-cells are the most abundant and functionally significant component of the pancreatic islets. These &#946;-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 &#946;-cells. This autoimmune attack is primarily mediated by pro-inflammatory cytokines, including interleukin-1&#946; (IL-1&#946;), tumor necrosis factor-alpha (TNF-&#945;), and interferon-gamma (IFN-&#947;)2. These cytokines initiate a cascade of signaling events within &#946;-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 &#946;-cell mass and function during the onset of T1D3.
Understanding the molecular mechanisms that drive &#946;-cell apoptosis is critical for identifying strategies to prevent or mitigate &#946;-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 &#946;-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 &#946;-cell dysfunction and death in vitro4,5. These experiments provide key insights into the vulnerability and survival of &#946;-cells under disease-associated conditions, and they also serve as a platform for testing therapeutic interventions aimed at protecting or rescuing &#946;-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-&#945;, 5 ng/mL IL-1&#946;, and 100 ng/mL IFN-&#947;) 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 &#946;-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 &#946;-cell death, specific dyes targeting only the insulin-producing cells are needed. A distinct feature of the pancreatic &#946;-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 &#946;-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 &#946;-cell viability and function11. UV probes like TSQ and Zinquin that are highly selective towards Zn2+ have been developed to quantify &#946;-cells by imaging and measurement of free intracellular Zn2+&#160;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 &#946;-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 &#946;-cell viability and apoptosis, which is critical for understanding and mitigating &#946;-cell destruction in diabetes research and drug development.

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

isolated pancreatic islet treatment and apoptosis measurement

Isolated Pancreatic Islet Treatment and Apoptosis Measurement

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing &#946;-cells, using a combination of fluorescence staining techniques and confocal microscopy to assess cell viability, apoptosis, and &#946;-cell-specific death. Isolated mouse islets were treated with varying concentrations of a cytokine cocktail, including TNF-&#945;, IL-1&#946;, and IFN-&#947;, 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 &#946;-cells, Zn2+ selective indicator staining was used to label insulin-producing cells through the zinc association in insulin granules, revealing substantial &#946;-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 &#946;-cell apoptosis in early type 1 diabetes.

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 co...

This study utilizes multi-staining fluorescence-based markers of cell death and apoptosis combined with confocal microscopy to assess cytokine-induced apoptosis and &#946;-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 &#946;-cells, using a combination of ...

isolated-pancreatic-islet-treatment-and-apoptosis-measurement

Research

JoVE Journal

Biology

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Isolation of Human Islets from Partially Pancreatectomized Patients

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. This protocol explains a method for isolating pancreatic islets by first perfusing collagenase via Ampulla of Vater to digest the exocrine pancreatic tissue and a simplified density gradient centrifugation to purify the islets from mice.

Pancreatic Islets Isolation through Collagenase Perfusion: An Enzymatic Method to Isolate Pancreatic Islet Cells

Assaying in vitro β-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining β-cell function.

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250–350 high quality and fully functional islets per mouse.

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

This protocol provides a step-by-step guide for the procurement of a porcine pancreas for islet isolation and purification.

Surgical Perfusion and Isolation of the Porcine Pancreas for Islet Isolation

Here, we provide details on performing glucose-stimulated insulin secretion (GSIS) via perfusion through the mice vasculature with an intact pancreas as an additional technique to assess the endocrine pancreatic function with reduction of possible confounders.

Glucose-Stimulated Insulin Secretion via Perfusion through the Mice Vasculature with an Intact Pancreas

This protocol describes a standardized method for surgical harvesting, enzymatic digestion, and density-gradient purification of Lewis rat pancreatic islets, enabling high-yield, high-purity preparations for direct use in transplantation or in vitro studies.

Pancreatic Islet Isolation and Purification from Lewis Rats Using Enzymatic Digestion and Density-Gradient Separation

Learn multi-staining fluorescence techniques to study apoptosis in mouse pancreatic islets under cytokine treatment. This method reveals spatial patterns of β-cell death relevant to type 1 diabetes research.

Isolated Pancreatic Islet Treatment and Apoptosis Measurement

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