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Biology

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Published: August 12th, 2018

DOI:

10.3791/58099

1Peripheral Neuropathy Research Center, College of Medicine, Dong-A University, 2Department of Biochemistry, College of Medicine, Dong-A University, 3Aging Institute, Department of Medicine, University of Pittsburgh School of Medicine
* These authors contributed equally

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitochondria are organelles that are essential for cell proliferation and physiology. Mitochondria are responsible for generating more than 80% of the ATP supply via oxidative phosphorylation, and they also provide various metabolic intermediates for biosynthesis and metabolism1,2. In addition to their roles in energy supply and metabolism, mitochondria play central roles in many other important processes, including reactive oxygen species (ROS) generation, the regulation of cell death, and intracellular Ca2+ dynamics3. Alterations in mitochondrial function have been associat....

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1. Generation of HeLa cells expressing mito-Keima (mt-Keima)

  1. Preparation of mt-Keima lentivirus
    1. Coat a 100-mm culture dish by adding 2 mL of 0.001% poly-L-lysine/phosphate-buffered saline (PBS) and allow it to stand for 5 min at room temperature.
    2. Remove the poly-L-lysine solution using a glass pipette connected to a vacuum and wash the culture dish by adding 2 mL of 1x PBS.
    3. Plate 1.5 x 106 HEK293T cells on the coated culture dish with 10 mL of DMEM.......

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An example of the flow cytometry analysis of CCCP-induced mitophagy in HeLa-Parkin cells is shown in Figure 4. Using the flow cytometry analysis method described above, we can detect a dramatic increase in mitophagic cells in the "high" gate. The percentage of cells in the "high" gate was increased more than 10-fold compared with untreated control cells (Figure 4A). This increase in mitophagy activity was complete.......

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Here, we present a rapid and sensitive method for using flow cytometry to measure cellular mitophagy activity in cells expressing mt-Keima. Cells undergoing a high level of mitophagy exhibit an increased ratio of PE-CF594 (561 nm)/BV605 (405 nm) excitation. Thus, mitophagy activity can be expressed as the percentage of cells exhibiting a high 561/405 ratio. We calculated the percentage of cells in the "high" gate region on a dot plot of PE-CF594 (561 nm) versus BV605 (405 nm), and the results showed that treatmen.......

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This work was supported by a grant from National Research Foundation of Korea (2016R1D1A1B03931949) (to J. U.), and by the National Research foundation of Korea (NRF) grant funded by the Korea government(MSIT) (No. 2016R1A2B2008887, No. 2016R1A5A2007009) (to J.Y.)

....

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Name Company Catalog Number Comments
REAGENTS
poly-L-lysine Sigma-Aldrich P2636
FBS GIBCO 16000-044
penicillin/streptomycin wellgene LS202-02
PBS Hyclone SH30013.02
HEK293T ATCC CRL-3216
DMEM GIBCO 12800-082
OPTI-MEM  GIBCO 31985-070
Turbofect Thermos scientific R0531
0.45 μm syringe filter sartorius 16555
HeLa ATCC CCL-2
polybrene Sigma-Aldrich H9268 8 mg/ml
puromycin Sigma-Aldrich P8833 2 mg/ml 
Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) Sigma-Aldrich C2759 10 mM
trypsin-EDTA wellgene LS015-01
EQUIPMENTS
BD LSRFortessa BD Bioscience LSRFortessa
FACSDIVA BD Bioscience FACSDIVA (v8.0.1)

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