Published: May 19th, 2023
Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.
Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes.
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.
Mitochondria are essential for all aerobic animals, including humans. They convert the chemical energy of biomolecules to adenosine triphosphate (ATP) via oxidative phosphorylation1, synthesize heme2, degrade fatty acids through β oxidation3, regulate calcium4 and iron5 homeostasis, control cell death by apoptosis6, and generate reactive oxygen species (ROS), which play a vital role in redox homeostasis7. Two complementary and opposite processes maintain the integrity and proper function o....
NOTE: For the convenience of the readers, we have divided the protocol into two parts: one focuses on the protocol for measuring mitophagy in C. elegans, and the other focuses on the protocol for measuring mitophagy in liver cells. The list of materials can be found in the Table of Materials provided.
1. The C. elegans protocol
Induction of a robust mitophagy response in both C. elegans worms and Hep-3B cells with VL-850
VL-850 protects C. elegans worms and human keratinocytes (HaCaT cells) from oxidative stress23. To further explore its mechanism of action, we examined whether VL-850 induces mitophagy in C. elegans and other human cells. To test this, we exposed C. elegans worms (young adults, 3 days post-L1) to 62.5 µM VL-850, 5 µM FCCP (positive cont.......
Multiple mitophagy pathways involve various proteins and biomolecules (e.g., cardiolipin29). However, the endpoint of these pathways is similar-the degradation of mitochondria by lysosomal enzymes12,13. Indeed, several methods use this endpoint to quantify mitophagy. However, some methods, such as electron microscopy, demand access to costly equipment, trained experts, and an extended preparation time for the specimens and analysis. Furthe.......
We thank members of the Gross laboratory for the critical reading of the manuscript and their comments and advice. We thank the Caenorhabditis Genetics Center (CGC), which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440), for providing some of the strains. This research was supported by a grant from Vitalunga Ltd and the Israel Science Foundation (grant No. 989/19). The graphical abstract figure (Figure 1) was generated with BioRender.com.....
|Reagent or resource
|Bacto Yeast extract
|Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)
|DMEM high glucose
|Double distilled water (DDW)
|Dulbecco's Phosphate Buffered Saline (PBS)
|FBS heat inactivated
|HEPES Buffer 1 M
|Lysosome/Mitochondria/Nuclear Staining Cytopainter Reagent
|Nonidet P 40
|Electron Microscopy Sciences
|Poloxamer 188 Solution
|Potassium dihydrogen phosphate
|Potassium phosphate dibasic
|SeaKem LE Agarose
|Sodium phosphate dibasic dodecahydrate
|0.22 μm syringe filter
|1.7 mL Micro Centrifuge Tubes
|10 cm Petri plates
|1,000 mL Erlenmeyer Flask
|15 mL Sterile Polypropylene tube
|35 mm Petri dishes
|500 mL vacuum filter/storage bottle system, 0.22 μm
|50 mL Sterile Polypropylene tube
|Deckgläser Microscope cover glass 24 x 60 mm
|Glass test tubes (10 mL- 13 x 100 mm) Borosilicate glass
|iBiDi 8 well μ-slides
|Microscope cover glass 24 x 40 mm
|Platinum iridium 0.25 mM wire
|World Precision Instruments
|Cell counter CellDrop BF
|Nikon Yokogawa W1 Spinning Disk confocal microscope with DAPI, FITC, and TRITC filters and bright-field, with a 60x CFI Plan-Apochromat Lambda type lens (air lens) and NIS-Elements software
|Olympus SZ61 stereo microscope
|Revolver Adjustable Lab Rotator
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