single-cell dissociation of murine islets for flow cytometric analysis of &#946;-cell mitophagy

Quantitative Analysis of Mitophagy in Murine Pancreatic &amp;#946;-Cells

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.

Author Spotlight: Assessment of Mitophagy Flux in Pancreatic &amp;#946;-Cells Using Effective and Robust Complementary Approaches 

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

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 Single-Cell Dissociation of Murine Islets for Flow Cytometric Analysis of β-Cell Mitophagy at JoVE.com

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.

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Research

JoVE Journal

Biology

90 次观看.  University of Michigan, Ann Arbor. 首先从常规脂肪饮食或高脂肪饮食小鼠中分离出的孔眼样品在37°C下培养过夜。为研究设计所需的实验条件。对于每个实验条件，使用移液管，将100个孔眼挑入含有两毫升孔孔培养基的六厘米培养皿中。为了诱导线粒体自噬，用两微升 250 纳摩尔缬氨霉素原液处理适当培养皿中的孔眼三小时。分离孔眼并使用移液管将孔眼从每个培养皿转移到单独的微量离心管中。然后，...