flow cytometric analysis of mitophagy in murine pancreatic &#946;-cells using the genetically encoded mt-keima reporter

Análisis cuantitativo de la mitofagia en células β pancreáticas murinas

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.

Autor destacado: Evaluación del flujo de mitofagia en las células β pancreáticas utilizando enfoques complementarios efectivos y robustos 

Enfoques complementarios para interrogar el flujo de mitofagia en las células β pancreáticas

Watch this Scientific Journal Video about Flow Cytometric Analysis of Mitophagy in Murine Pancreatic Î²-Cells Using the Genetically Encoded mt-Keima Reporter at JoVE.com

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

Análisis citométrico de flujo de la mitofagia en células β pancreáticas murinas utilizando el mt-Keima Reporter codificado genéticamente

Para evaluar la mitofagia en células beta pancreáticas murinas utilizando el indicador de mitofagia codificado genéticamente, se cultivaron islotes pancreáticos aislados de ratones de tipo salvaje y Mt-Keima durante la noche a 37 grados centígrados. Al día siguiente, agregue 100 islotes en una placa de Petri de seis centímetros que contenga dos mililitros de medio de islote para cada condición experimental utilizada en este protocolo. A continuación, disociar los ojales en células individuales y teñirlos con FluoZin-3-AM y DAPI como se demostró anteriormente para el enfoque basado en el colorante Mt-Phagy.A continuación, proceda con el análisis citométrico de flujo de las muestras. Ajuste los voltajes de dispersión directa o FSC y dispersión lateral o SSC para lograr una distribución uniforme de celdas en un gráfico SSCA versus FSCA. Para seleccionar celdas individuales y excluir múltiples, agregue una puerta rectangular en la altura FSC frente a la anchura FSC, seguida de la altura SSC frente a la anchura SSE.Los multipletes se excluyen debido a sus valores de señal de ancho más altos. A continuación, configure una puerta negativa DAPI para excluir las células muertas. Después de eso, configure las puertas positivas de FluoZin-3-AM para filtrar las células beta vivas.Establezca un esquema de compuerta triangular utilizando la muestra positiva de Mt-Keima para identificar poblaciones de células ácidas y neutras. Una vez establecidas las puertas primaria y de fluorescencia, se evaluó el flujo de mitofagia utilizando cambios basales e inducidos por valinomicina en la fluorescencia de Mt-Keima.

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Biology

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

84 vistas. Para evaluar la mitofagia en células beta pancreáticas murinas utilizando el indicador de mitofagia codificado genéticamente, se cultivaron islotes pancreáticos aislados de ratones de tipo salvaje y Mt-Keima durante la noche a 37 grados centígrados. Al día siguiente, agregue 100 islotes en una placa de Petri de seis centímetros que contenga dos mililitros de medio de islote para cada condición experimental utilizada en este protocolo. A continuación, disociar los ojales en células i...

Video: Flow Cytometric Analysis of Mitophagy in Murine Pancreatic β-Cells Using the Genetically Encoded mt-Keima Reporter  

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 ...