Mito-kaede photoactivation and chase experiment for mitophagy: optimizing flux measurement via fluid exchange system.

Modulating autophagy and mitophagy, vital cellular quality control systems, offer therapeutic potential for critical illnesses. However, limited drug screening options hinder progress. We present a novel assay using the photoswitchable fluorescent reporter, mito-Kaede, to quantify mitophagy flux. Mito-Kaede's superior UV-induced photoconversion and brightness post-conversion make it ideal for prolonged mitochondrial dynamics tracking. Its specificity in responding to mitophagy, confirmed by parkin-knockout cells, adds value. When coupled with a custom fluid exchange system, enabling efficient medium changes, precise mitophagy observations become feasible. This mitophagy assay, alongside our methodological insights, can decipher mitophagy's role in pathology and supports drug screening efforts.