Name: 作者聚焦：使用有效且稳健的互补方法评估胰腺 β 细胞中的线粒体自噬通量
Uploaded: 2023-09-15T14:40:00
Description: Watch this Scientific Journal Video about Assessment of Mitophagy Flux in Pancreatic β-Cells Using Effective and Robust Complementary Approaches at JoVE.com

E.L-D.感谢 NIH（T32-AI007413 和 T32-AG000114）的支持。SAS 感谢 JDRF （COE-2019-861）、NIH（R01 DK135268、R01 DK108921、R01 DK135032、R01 DK136547、U01 DK127747）、退伍军人事务部 （I01 BX004444）、Brehm 家族和 Anthony 家族的支持。

本协议中提出的动物研究已由密歇根大学机构动物护理和使用委员会审查和批准。本研究使用20周龄的雄性C57BL / 6J小鼠，采用15周的常规脂肪饮食（RFD）或高脂肪饮食（HFD）。
1. 通过基于染料的 MtPhagy 方法评估线粒体自噬（方法 1）
<ol><li>小鼠胰岛的制备和处理<ol><li>按照以下步骤进行胰岛培养和缬氨霉素暴露。<ol><li>按照先前描述的方法2,1...

小鼠胰腺 β 细胞中线粒体自噬的定量分析

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该协议描述了两种互补的方法来量化解离的原代小鼠胰岛中的线粒体自噬通量。使用 mt-Keima 方法，线粒体自噬的增加被量化为酸性 （561 nm）/中性 （405 nm） 细胞比率的增加，而在 MtPhagy 方法中，线粒体自噬通量的增加被量化为 Fluozin高MtPhagy高TMRE低 细胞群的增加。这些方法可以对线粒体自噬通量进行快速、定量和β细胞特异性评估。
这两种方法都是?...

胰腺β细胞产生和分泌胰岛素以满足代谢需求，β细胞功能障碍是 1 型和 2 型糖尿病高血糖和糖尿病发病的原因。β细胞通过线粒体能量和代谢输出将葡萄糖代谢与胰岛素分泌结合，这取决于功能性线粒体质量的储备 1,2,3。为了保持最佳的β细胞功能，β细胞依靠线粒体质量控制机制来去除老化或受损的线粒体并保留功能性线粒体质量4。选择性线粒体自噬，也称为线粒体自噬，是线粒体质量控制途径的关键组成部分。
活细胞中线粒体自噬的评估通常依赖于线粒体自噬期间发生的线粒体 pH 值的变化。线粒体的 pH 值呈微碱性，健康的线粒体通常位于 pH 值中性的胞质溶胶中。在线粒体自噬过程中，受损或功能失调的线粒体被选择性地掺入自噬体中，并最终在酸性溶酶体中清除5.几种体内转基因线粒自噬报告小鼠模型，如 mt-Keima6、mitoQC7 和 CMMR8，以及可转染的线粒自噬探针，如 Cox8-EGFP-mCherry 质粒9，利用这种 pH 变化来提供线粒自噬的定量评估。使用表达 mt-Keima pH 敏感双激发比率探针的转基因小鼠已通过流式细胞术10,11 优化用于胰岛和β细胞的线粒体自噬评估。酸性与中性 mt-Keima 波长发射的比率（酸性 561 nm 与中性 480 nm 激发的比率）可用于稳健地量化线粒体自噬 6,12。
该协议描述了一种优化的方法来评估从mt-Keima转基因小鼠10,11分离的原代胰岛和β细胞中的线粒体自噬通量。虽然 mt-Keima 是一种高度灵敏的探针，但它需要复杂的动物育种方案或细胞转染，这在与其他遗传模型或原代人类胰岛结合使用时通常具有挑战性。此外，使用多种荧光激光器和检测器来识别中性和酸性细胞群可能会限制其他荧光报告基因的组合使用。
为了克服这些挑战，该协议还描述了一种互补的，单荧光通道，基于染料的方法，用于从分离的小鼠胰岛中稳健检测β细胞中的线粒体自噬。这种方法被称为 MtPhagy 方法，它利用三种细胞渗透性染料的组合来选择β细胞，量化积极经历线粒体自噬的细胞群，并同时评估线粒体膜电位（MMP 或 Δψm）。
这些染料中的第一种是 Fluozin-3-AM，这是一种细胞渗透性 Zn2+ 指示剂，Ex/Em 为 494/516 nm13。小鼠胰岛由功能不同的细胞组成异质性细胞群，包括 α、β、δ 和 PP 细胞。β细胞约占小鼠胰岛细胞的 80%，由于其在胰岛素颗粒中的高 Zn2+ 浓度14,15，因此可以与其他胰岛细胞类型区分开来，从而可以将β细胞鉴定为 Fluozin-3-AM高细胞群。MtPhagy 染料是一种 pH 敏感染料，通过化学键固定在线粒体上并发出弱荧光，也用于该协议16。线粒体自噬诱导后，受损的线粒体被掺入酸性溶酶体中，MtPhagy 染料在低 pH 环境 （Ex/Em 561/570-700 nm） 内增加其荧光强度。
此外，四甲基罗丹明乙酯 （TMRE） 用于评估 MMP。TMRE 是一种细胞渗透性带正电荷的染料 （Ex/Em 552/575 nm），由于其膜电位17 所支撑的相对负电荷，它被健康的线粒体隔离。受损或不健康的线粒体会耗散其膜电位，导致螯合 TMRE 的能力降低。将这些染料一起使用，通过流式细胞术可以将进行线粒体自噬的β细胞鉴定为 Fluozin高MtPhagy高TMRE低 群体。由于线粒体自噬是一个动态而不是静态过程，因此该方案进行了优化，以使用缬氨霉素评估线粒体自噬通量，缬氨霉素是一种 K+ 离子载体，可在 MMP18 消散后诱导线粒体自噬。在存在和不存在缬氨霉素的情况下比较线粒体自噬可以评估不同样品组的线粒体自噬通量。
与mt-Keima方案不同，当前方法的基于染料的性质使其可以外推到人类胰岛和其他难以转染的细胞类型，并避免了对复杂动物育种方案的需求。该协议的总体目标是通过两种独立的基于流式细胞术的方法在单细胞水平上量化β细胞中的线粒体自噬。综上所述，该协议描述了两种强大且互补的方法，它们允许在线粒体质量控制的定量研究中具有精确性和灵活性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT Flow Cytometer</td>
			<td>Thermofisher Scientific</td>
			<td>A24858</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermofisher Scientific</td>
			<td>D1306</td>
			<td>DAPI reconstituted in ddH2O to reach 0.2 &#181;g/mL stock</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>317275</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty Acid Free heat shock BSA powder</td>
			<td>Equitech</td>
			<td>BAH66</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gemini Bio</td>
			<td>900-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluozin-3AM&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>F24195</td>
			<td>100 &#956;g Fluozin-3AM powder reconstituted in 51 &#956;L DMSO and 51 &#956;L Pluronic F-127 to reach 1 mM stock.&#160;</td>
		</tr>
		<tr>
			<td>Gibco&#160;RPMI 1640 Medium</td>
			<td>Fisher Scientific</td>
			<td>11-875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>MtPhagy dye</td>
			<td>Dojindo</td>
			<td>MT02-10</td>
			<td>5 &#956;g MtPhagy powder reconstituted with 50 &#956;L DMSO to reach 100 &#956;M stock.&#160;</td>
		</tr>
		<tr>
			<td>MtPhagy dye</td>
			<td>Dojindo</td>
			<td>MT02-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100x)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>1x Solution used in procotol by diluting 1:10 in ddH2O</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, 10x</td>
			<td>Fisher Scientific</td>
			<td>BP399-20</td>
			<td>1x Solution used in procotol by diluting 1:10 in ddH2O</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100x)</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td>5 &#956;g MtPhagy powder reconstituted with 50 &#956;L DMSO to reach 100 &#956;M stock.&#160;</td>
		</tr>
		<tr>
			<td>TMRE [Tetramethylrhodamine, ethyl ester, perchlorate]</td>
			<td>Anaspec</td>
			<td>AS-88061</td>
			<td>TMRE powder reconstituted in DMSO to reach 100 &#956;M stock.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermofisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Valinomycin</td>
			<td>Sigma</td>
			<td>V0627</td>
			<td>Valinomycin powder reconsituted in DMSO to reach 250 nM stock.</td>
		</tr>
	</tbody>
</table>

complementary approaches to interrogate mitophagy flux in pancreatic &#946;-cells

线粒体自噬是维持最佳线粒体功能所必需的质量控制机制。功能失调的β细胞线粒体自噬导致胰岛素释放不足。线粒体自噬的高级定量评估通常需要使用遗传报告基因。mt-Keima 小鼠模型表达了一种线粒体靶向 pH 敏感的双激发比率探针，用于通过流式细胞术定量线粒体自噬，已在β细胞中进行了优化。酸性与中性 mt-Keima 波长发射的比率可用于稳健地量化线粒体自噬。然而，在处理复杂的遗传小鼠模型或难以转染的细胞（如原代人类胰岛）时，使用遗传线粒体自噬报告基因可能具有挑战性。该协议描述了一种基于互补染料的新型方法，用于使用MtPhagy量化原代胰岛中的β细胞线粒体自噬。MtPhagy 是一种对 pH 敏感的细胞渗透性染料，当线粒体处于低 pH 值环境中时，例如线粒体自噬期间的溶酶体，它会积聚在线粒体中并增加其荧光强度。通过将 MtPhagy 染料与 Fluozin-3-AM（一种选择β细胞的 Zn2+ 指示剂）和四甲基罗丹明乙酯 （TMRE） 相结合来评估线粒体膜电位，可以通过流式细胞术特异性定量β细胞中的线粒体自噬通量。这两种方法具有高度互补性，可以在许多β细胞模型中评估线粒体质量控制时具有灵活性和精确性。

Pancreatic &#946;-cells produce and secrete insulin to meet metabolic demands, and &#946;-cell dysfunction is responsible for hyperglycemia and diabetes onset in both type 1 and type 2 diabetes. &#946;-Cells couple glucose metabolism with insulin secretion via mitochondrial energetics and metabolic output, which depend on a reserve of functional mitochondrial mass1,2,3. To maintain optimal &#946;-cell function, &#946;-cells rely on mitochondrial quality control mechanisms to remove aged or damaged mitochondria and preserve functional mitochondrial mass4. Selective mitochondrial autophagy, also known as mitophagy, is a key component of the mitochondrial quality control pathway.
Assessments of mitophagy in live cells often rely on changes in mitochondrial pH that occur during mitophagy. Mitochondria have a slightly alkaline pH, and healthy mitochondria normally reside in the pH-neutral cytosol. During mitophagy, damaged or dysfunctional mitochondria are selectively incorporated into autophagosomes and eventually cleared within acidic lysosomes5. Several in vivo transgenic mitophagy reporter mouse models, such as mt-Keima6, mitoQC7, and CMMR8, as well as transfectable mitophagy probes, such as the Cox8-EGFP-mCherry plasmid9, utilize this pH change to provide quantitative assessments of mitophagy. Use of transgenic mice expressing the mt-Keima pH-sensitive dual-excitation ratiometric probe has been optimized for mitophagy assessments in islets and &#946;-cells via flow cytometry10,11. The ratio of acidic-to-neutral mt-Keima wavelength emissions (the ratio of acidic 561 nm to neutral 480 nm excitation) can be used to robustly quantify mitophagy6,12.
This protocol describes an optimized approach to assess mitophagy flux in primary islets and &#946;-cells isolated from mt-Keima transgenic mice10,11. While mt-Keima is a highly sensitive probe, it requires either complicated animal breeding schemes or the transfection of cells, which can often be challenging when working in combination with other genetic models or with primary human islets. Additionally, the use of multiple fluorescence lasers and detectors to identify neutral and acidic cell populations can limit the combinatorial use of other fluorescent reporters.
To overcome these challenges, this protocol also describes a complementary, single fluorescent channel, dye-based method for robust detection of mitophagy in &#946;-cells from isolated mouse islets. This approach, referred to as the MtPhagy method, utilizes a combination of three cell-permeable dyes to select for &#946;-cells, quantify the cell populations actively undergoing mitophagy, and assess mitochondrial membrane potential (MMP or &#916;&#968;m) simultaneously.
The first of these dyes is Fluozin-3-AM, a cell-permeable Zn2+ indicator with an Ex/Em 494/516 nm13. Mouse islets comprise a heterogeneous population of functionally distinct cells, including &#945;-, &#946;-, &#948;-, and PP cells. &#946;-Cells comprise approximately 80% of cells within the mouse islet and can be distinguished from other islet cell types due to their high Zn2+ concentration within insulin granules14,15, allowing for identification of &#946;-cells as the Fluozin-3-AMhigh population. The MtPhagy dye, a pH-sensitive dye that is immobilized on mitochondria via a chemical bond and emits weak fluorescence, is also utilized in this protocol16. Upon mitophagy induction, damaged mitochondria are incorporated into the acidic lysosome, and the MtPhagy dye increases its fluorescence intensity within the low pH environment (Ex/Em 561/570-700 nm).
Additionally, Tetramethylrhodamine, ethyl ester (TMRE), is used to assess MMP. TMRE is a cell-permeable positively charged dye (Ex/Em 552/575 nm) that is sequestered by healthy mitochondria due to the relative negative charge upheld by their membrane potential17. Damaged or unhealthy mitochondria dissipate their membrane potential, resulting in decreased ability to sequester TMRE. Utilizing these dyes together, &#946;-cells undergoing mitophagy can be identified as the FluozinhighMtPhagyhighTMRElow population via flow cytometry. Since mitophagy is a dynamic rather than static process, this protocol was optimized to assess mitophagy flux using valinomycin, a K+-ionophore that induces mitophagy following dissipation of MMP18. Comparison of mitophagy in the presence and absence of valinomycin allows for assessment of mitophagy flux in different sample groups.
The dye-based nature of the current approach allows it to be extrapolated to human islets and other difficult-to-transfect cell types and circumvents the need for complicated animal breeding schemes, unlike the mt-Keima protocol. The overarching goal of this protocol is to quantify mitophagy in &#946;-cells at the single-cell level via two independent flow cytometry-based methods. Taken together, this protocol describes two powerful and complementary methods that allow for both precision and flexibility in the quantitative study of mitochondrial quality control.

作者聚焦：使用有效且稳健的互补方法评估胰腺 β 细胞中的线粒体自噬通量 

研究胰腺β细胞线粒体自噬通量的互补方法

Our protocol describes two complementary methods to study beta-cell mitophagy flux at the single-cell resolution via flow cytometry. The mt-Keima protocol utilizes a genetic mitophagy reporter, whereas the MtPhagy protocol combines three fluorescent dyes, which makes it easy to extrapolate to difficult-to-transfect cells in human samples. Assessments of mitophagy using traditional biochemical approaches and imaging approaches are both time consuming and challenging.Thus, it's important to develop new, robust, and effective methods to study mitophagy flux in beta-cells. Both the mt-Keima and MtPhagy method are quantitative assessments that are efficient for studying mitophagy flux in pancreatic beta cells. So, the Soleimanpour lab focuses on the different mechanisms that drive pancreatic beta-cell failure and diabetes.And we specifically focus on the different aspects of mitochondrial lifecycle, including mitophagy, to understand how defects in mitochondrial function drive the development of diabetes.

通过基于染料的 MtPhagy 方法评估线粒体自噬 这种基于染料的方法经过优化，无需遗传报告基因即可分析原代小鼠β细胞内的线粒体自噬通量，使用 Fluozin-3-AM、TMRE 和 MtPhagy 以及 DAPI 排除死细胞。通过将这些染料与缬氨霉素配对以诱导线粒体自噬，该方案概述了一种基于染料的方法，以选择性地测量原代小鼠β细胞中的线粒体自噬通量18。对于使用这种 MtPhagy 方法显?...

Watch this Scientific Journal Video about Assessment of Mitophagy Flux in Pancreatic β-Cells Using Effective and Robust Complementary Approaches at JoVE.com

该协议概述了对胰腺β细胞中线粒体自噬进行定量分析的两种方法：第一种是细胞渗透性线粒体特异性染料的组合，第二种是遗传编码的线粒体自噬报告基因。这两种技术是互补的，可以根据特定需求进行部署，从而在定量解决线粒体质量控制问题时具有灵活性和精确性。

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

我们的方案描述了两种互补的方法，通过流式细胞术在单细胞分辨率下研究β细胞线粒体自噬通量。mt-Keima 方案利用遗传线粒体自噬报告基因，而 MtPhagy 方案结合了三种荧光染料，这使得很容易推断到人类样本中难以转染的细胞。使用传统的生化方法和成像方法评估线粒体自噬既耗时又具有挑战性。因此，开发新的、稳健的和有效的方法来研究β细胞中的线粒体自噬通量是很重要的。mt-Keima 和 MtPhagy 方法都是定量评估，可有效研究胰腺 β 细胞中的线粒体自噬通量。因此，Soleimanpour 实验室专注于驱动胰腺 β 细胞衰竭和糖尿病的不同机制。我们特别关注线粒体生命周期的不同方面，包括线粒体自噬，以了解线粒体功能缺陷如何驱动糖尿病的发展。

complementary-approaches-to-interrogate-mitophagy-flux-pancreatic

Research

JoVE Journal

Biology

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

科研

生物学

Single-Cell Dissociation of Murine Islets for Flow Cytometric Analysis of &#946;-Cell Mitophagy

Begin by culturing the eyelet samples isolated from regular fat diet or high fat diet mice overnight at 37 degrees Celsius. Design the desired experimental conditions for the study. For each experimental condition, using a pipette, pick 100 eyelets into a six centimeter Petri dish containing two milliliters of eyelet medium.To induce Mitophagy, treat the eyelets in the appropriate Petri dishes with two microliters of 250 nano molar Valinomycin stock for three hours. Isolate the eyelets and using a pipette, transfer the eyelets from each Petri dish to a separate micro centrifuge tube. Then, spin the eyelets at 350 G for one minute at 10 degrees Celsius and using a pipette, discard the supernatant.Wash the obtained sample twice with one milliliter of PBS. Between the washes, spin the sample and discard the supernatant as demonstrated previously. To dissociate eyelets into single cells, add 500 microliters of 0.05%Trypsin prewarmed to 37 degrees celsius, to one sample tube.Pipette the contents of the tube up and down repeatedly until the eyelets are visibly dispersed. Immediately add one milliliter of prewarmed eyelet medium to neutralize trypsin. Spin the samples at 500 G for five minutes at 10 degrees Celsius.Remove the supernatant carefully without disturbing the pellet. Then, wash the samples two times with eyelet flow medium. The cell samples are now ready to be stained for flow cytometric analysis of beta cell mitophagy.

小鼠胰岛的单细胞解离用于β细胞线粒体自噬的流式细胞术分析

Flow Cytometric Analysis of Mitophagy in Murine Pancreatic &#946;-Cells Using MtPhagy Dye

Proceed to perform flow cytometric analysis of beta cell mitophagy, with a single cell suspension of about 100 isolated mirroring islets prepared, corresponding to each desired experimental condition to be studied. First, resuspend the islet samples for each condition in 500 microliters of islet flow medium. Add 0.25 microliters of MtPhagy stock to tubes designated to receive the MtPhagy dye.Similarly, add 0.25 microliters of TMRE stock and fluozin-3-AM stock to the respective designated tubes. Wrap the tubes in aluminum foil and vortex each tube at low speed for five seconds to mix the contents. Then incubate the tubes at 37 degrees Celsius for 30 minutes.Halfway through incubation, vortex each sample at low speed for five to 10 seconds. After incubation, centrifuge the tubes at 350 G for one minute at 10 degrees Celsius. Discard the supernatant.And resuspend the samples in 500 microliters of islet flow medium. Add DAPI to the microcentrifuge tubes containing samples that require DAPI treatment. Centrifuge again and discard the supernatant as demonstrated previously.Resuspend the residue in 500 microliters of islet flow medium. Finally, place the samples on ice and then proceed with flow cytometry. Adjust the voltages for forward scatter or FSC and side scatter or SSC to ensure cell populations are at the center of the scatter plot.To exclude multiplets, at a rectangular gate on FSC height versus FSC width, followed by SSC height versus SSC width. Adjust voltages and compensation for DAPI to filter for live beta cells. Set fluorescence gates for each fluorophore used based on the unstained RFD sample.Once gates are established, collect 10, 000 events per sample. Beta cells with high utilization of mitophagy were defined as the fluozin high MtPhagy high TMRE low population in quadrant three or Q3.Basal and valinomycin-induced mitophagy levels were characterized in both RFD and HFD islets.

使用 MtPhagy 染料对小鼠胰腺β细胞中的线粒体自噬进行流式细胞术分析

Flow Cytometric Analysis of Mitophagy in Murine Pancreatic &#946;-Cells Using the Genetically Encoded mt-Keima Reporter

To assess mitophagy in murine pancreatic beta cells using the genetically encoded mitophagy reporter, culture pancreatic islets isolated from wild type and Mt-Keima mice overnight at 37 degrees Celsius. The following day, add 100 islets into a six centimeter Petri dish containing two milliliters of islet medium for each experimental condition used in this protocol. Next, to dissociate the eyelets into single cells and stain with FluoZin-3-AM and DAPI as demonstrated previously for the Mt-Phagy dye based approach.Then proceed with the flow cytometric analysis of the samples. Adjust the forward scatter or FSC and side scatter or SSC voltages to attain an even distribution of cells on an SSCA versus FSCA plot. To select single cells and exclude multiplets, add a rectangular gate on FSC height versus FSC width, followed by SSC height versus SSE width.The multiplets are excluded due to their higher width signal values. Then set up a DAPI negative gate to exclude dead cells. After that, set up FluoZin-3-AM positive gates to filter for live beta cells.Set up a triangle gating scheme using the Mt-Keima positive sample to identify acidic and neutral cell populations. Once the primary and fluorescence gates were established, assess mitophagy flux using basal and valinomycin-induced changes in Mt-Keima fluorescence.