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

Kwantitatieve analyse van mitofagie in β-cellen van de pancreas van muizen

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.

Auteur in de schijnwerpers: beoordeling van mitofagieflux in pancreas-β-cellen met behulp van effectieve en robuuste complementaire benaderingen 

Complementaire benaderingen om mitofagieflux in pancreas-β-cellen te ondervragen

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  

Flowcytometrische analyse van mitofagie in β-cellen van de pancreas van muizen met behulp van MtPhagy-kleurstof

Ga verder met het uitvoeren van flowcytometrische analyse van bètacelmitofagie, met een eencellige suspensie van ongeveer 100 geïsoleerde spiegeleilandjes bereid, overeenkomend met elke gewenste experimentele conditie die moet worden bestudeerd. Resuspendeer eerst de eilandjesmonsters voor elke toestand in 500 microliter eilandjesstroommedium. Voeg 0,25 microliter MtPhagy-bouillon toe aan buizen die zijn aangewezen om de MtPhagy-kleurstof te ontvangen.Voeg op dezelfde manier 0,25 microliter TMRE-bouillon en fluozin-3-AM-bouillon toe aan de respectievelijke aangewezen buizen. Wikkel de buizen in aluminiumfolie en draai elke buis vijf seconden op lage snelheid om de inhoud te mengen. Incubeer de buisjes vervolgens 30 minuten bij 37 graden Celsius.Halverwege de incubatie, draai elk monster op lage snelheid gedurende vijf tot 10 seconden. Centrifugeer de buisjes na incubatie gedurende één minuut bij 350 G bij 10 graden Celsius. Gooi de supernatant weg.En resuspendeer de monsters in 500 microliter eilandjesstroommedium. Voeg DAPI toe aan de microcentrifugebuisjes met monsters die een DAPI-behandeling nodig hebben. Centrifugeer opnieuw en gooi het supernatans weg zoals eerder gedemonstreerd.Resuspendeer het residu in 500 microliter eilandjesstroommedium. Plaats ten slotte de monsters op ijs en ga vervolgens verder met flowcytometrie. Pas de spanningen aan voor voorwaartse verstrooiing of FSC en zijverstrooiing of SSC om ervoor te zorgen dat celpopulaties zich in het midden van het verstrooiingsdiagram bevinden.Om multiplets uit te sluiten, bij een rechthoekige poort op FSC-hoogte versus FSC-breedte, gevolgd door SSC-hoogte versus SSC-breedte. Pas de spanningen en compensatie voor DAPI aan om te filteren op levende bètacellen. Stel fluorescentiepoorten in voor elke fluorofoor die wordt gebruikt op basis van het ongekleurde RFD-monster.Zodra de poorten zijn vastgesteld, verzamelt u 10.000 gebeurtenissen per monster. Bètacellen met een hoog gebruik van mitofagie werden gedefinieerd als de fluozine-hoge MtPhagy hoge TMRE-lage populatie in kwadrant drie of Q3. Basale en valinomycine-geïnduceerde mitofagieniveaus werden gekarakteriseerd in zowel RFD- als HFD-eilandjes.

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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 ...