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

Kvantitativ analyse af mitofagi i β-celler fra murine bugspytkirtel

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.

Forfatter Spotlight: Vurdering af mitofagiflux i β-celler i bugspytkirtlen ved hjælp af effektive og robuste komplementære tilgange 

Komplementære tilgange til at forhøre mitofagiflux i β-celler i bugspytkirtlen

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  

Flow cytometrisk analyse af mitofagi, i murin pancreas β-celler ved anvendelse af MtPhagy farvestof

Fortsæt med at udføre flowcytometrisk analyse af betacellemitofagi, med en enkelt cellesuspension på ca. 100 isolerede spejløer forberedt, svarende til hver ønsket eksperimentel tilstand, der skal undersøges. Først resuspenderes øprøverne for hver tilstand i 500 mikroliter øflowmedium. Tilsæt 0,25 mikroliter MtPhagy lager til rør, der er udpeget til at modtage MtPhagy farvestoffet.På samme måde tilsættes 0.25 mikroliter TMRE-lager og fluozin-3-AM-lager til de respektive udpegede rør. Pak rørene ind i aluminiumsfolie og hvirvel hvert rør ved lav hastighed i fem sekunder for at blande indholdet. Derefter inkuberes rørene ved 37 grader Celsius i 30 minutter.Halvvejs gennem inkubation, hvirvel hver prøve ved lav hastighed i fem til 10 sekunder. Efter inkubation centrifugeres rørene ved 350 G i et minut ved 10 grader Celsius. Supernatanten kasseres.Og resuspender prøverne i 500 mikroliter øflowmedium. Tilsæt DAPI til mikrocentrifugeglassene, der indeholder prøver, der kræver DAPI-behandling. Supernatanten centrifugeres igen og kasseres som tidligere påvist.Resuspender resten i 500 mikroliter øflowmedium. Til sidst placeres prøverne på is og fortsæt derefter med flowcytometri. Juster spændingerne for fremadrettet spredning eller FSC og sidespredning eller SSC for at sikre, at cellepopulationer er i centrum af spredningsplottet.For at udelukke multipleter ved en rektangulær port på FSC-højde versus FSC-bredde efterfulgt af SSC-højde versus SSC-bredde. Juster spændinger og kompensation for DAPI for at filtrere efter levende betaceller. Indstil fluorescensporte for hver anvendt fluorofor baseret på den ufarvede RFD-prøve.Når porte er etableret, skal du indsamle 10.000 begivenheder pr. Prøve. Betaceller med høj udnyttelse af mitofagi, blev defineret som fluozin høj MtPhagy høj TMRE lav befolkning i kvadrant tre eller Q3. Basal og valinomycin-induceret mitofaginiveauer blev karakteriseret i både RFD- og HFD-øer.

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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 ...