Methods for Monitoring Mitophagy Using mt-Keima

Area of Science:

• Cell Biology
• Mitochondrial Research
• Autophagy

Background:

• Mitophagy, the selective degradation of damaged mitochondria, is crucial for cellular health.
• Accurate quantification of mitophagy is essential for understanding its role in various physiological and pathological processes.
• Existing methods for mitophagy assessment can be limited in scope or applicability to live-cell imaging.

Purpose of the Study:

• To describe detailed procedures for quantifying mitophagy using mitochondria-targeted Keima (mt-Keima).
• To enable ratiometric measurement of mitophagy in live cells.
• To provide protocols for both flow cytometry and live-cell confocal microscopy applications.

Main Methods:

• Utilizing mt-Keima, a pH-sensitive and acid-stable fluorescent protein with bimodal excitation.
• Calculating a ratiometric signal based on excitation peaks at 440 nm (neutral) and 586 nm (acidic).
• Applying flow cytometry and live-cell confocal microscopy for mitophagy quantification.

Main Results:

• Established protocols for measuring mitophagy using mt-Keima.
• Demonstrated the feasibility of ratiometric signal calculation for mitophagy assessment.
• Enabled mitophagy quantification in live cells via advanced imaging techniques.

Conclusions:

• mt-Keima serves as a reliable fluorescent probe for quantifying mitophagy.
• The described methods facilitate robust mitophagy measurements in live cellular models.
• This approach enhances the study of mitochondrial dynamics and cellular quality control mechanisms.