Name: Flow Cytometric Analysis of Mitophagy in Murine Pancreatic &#946;-Cells Using MtPhagy Dye
Uploaded: 2023-09-15T14:40:00
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Mitophagy in Murine Pancreatic β-Cells Using MtPhagy Dye at JoVE.com

flow cytometric analysis of mitophagy in murine pancreatic &#946;-cells using mtphagy dye

쥐 췌장 β세포의 유사분열(Mitophagy)에 대한 정량적 분석

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.

저자 스포트라이트: 효과적이고 강력한 보완적 접근법을 사용한 췌장 β세포의 미토파지 플럭스 평가 

췌장 β세포에서 미토파지 플럭스를 조사하기 위한 보완적 접근법

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.

Watch this Scientific Journal Video about Flow Cytometric Analysis of Mitophagy in Murine Pancreatic β-Cells Using MtPhagy Dye at JoVE.com

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

MtPhagy 염료를 사용한 쥐 췌장 β세포의 미토파지의 유세포 분석

연구하고자 하는 각 실험 조건에 대응하여 약 100개의 분리된 미러링 섬(mirroring islet)의 단일 세포 현탁액을 준비하여 베타 세포 미토파지(mitophagy)의 유세포 분석을 수행합니다. 먼저, 각 조건에 대한 섬 샘플을 500 마이크로리터의 섬 유동 매체에 재현탁시킵니다. MtPhagy 염료를 받도록 지정된 튜브에 0.25마이크로리터의 MtPhagy 스톡을 추가합니다.마찬가지로, 0.25 마이크로리터의 TMRE 스톡과 플루오진-3-AM 스톡을 각각의 지정된 튜브에 추가합니다. 튜브를 알루미늄 호일로 감싸고 각 튜브를 5초 동안 저속으로 소용돌이쳐 내용물을 섞습니다. 그런 다음 튜브를 섭씨 37도에서 30분 동안 배양합니다.배양 중간에 각 샘플을 5-10초 동안 저속으로 소용돌이칩니다. 배양 후 튜브를 섭씨 10도에서 350G에서 1분 동안 원심분리합니다. 상층액을 버리십시오.그리고 500 마이크로 리터의 섬 유동 매체에 샘플을 재현탁시킵니다. DAPI 처리가 필요한 샘플이 들어 있는 마이크로 원심분리기 튜브에 DAPI를 추가합니다. 다시 원심분리하고 이전에 설명한 대로 상층액을 버립니다.500 마이크로리터의 섬 유동 매체에 잔류물을 재현탁시킵니다. 마지막으로 샘플을 얼음 위에 놓고 유세포 분석을 진행합니다. 전방 산란 또는 FSC 및 측면 산란 또는 SSC에 대한 전압을 조정하여 세포 집단이 산점도 플롯의 중심에 오도록 합니다.배수를 제외하려면 FSC 높이 대 FSC 너비의 직사각형 게이트에서 SSC 높이 대 SSC 너비가 뒤따릅니다. DAPI에 대한 전압 및 보정을 조정하여 라이브 베타 세포를 필터링합니다. 염색되지 않은 RFD 샘플을 기반으로 사용되는 각 형광단에 대한 형광 게이트를 설정합니다.일단 문이 설치되면, 표본 당 10 의 000의 사건을 모으십시오. 미토파지의 이용률이 높은 베타 세포는 3사분면 또는 Q3에서 플루오진이 높은 MtPhagy, 높은 TMRE, 낮은 집단으로 정의되었습니다. 기초 및 발리노마이신 유도 미토파지 수준은 RFD 및 HFD 섬 모두에서 특성화되었습니다.

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JoVE Journal

Biology

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

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.

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

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생물학

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.

β세포 미토파지의 유세포 분석을 위한 쥐 섬의 단일 세포 해리

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.

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.