Quantitative assessment of mitophagy in irradiated cancer cells

Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through lysosomal degradation. Dysfunctional mitochondria are prone to release potentially cytotoxic factors including reactive oxygen species (ROS) and caspase activators, such as cytochrome c, somatic (CYCS). Thus, proficient mitophagic responses mediate prominent cytoprotective functions. Moreover, the rapid degradation of permeabilized mitochondria limits the release of mitochondrial components that may drive inflammatory reactions, such as mitochondrial DNA (mtDNA) and transcription factor A, mitochondrial (TFAM), implying that mitophagy also mediates potent anti-inflammatory effects. Here, we detail a simple, flow cytometry-assisted protocol for the specific measurement of mitophagic responses as driven by radiation therapy (RT) in mouse hormone receptor (HR)⁺ mammary carcinoma TS/A cells. With some variations, this method – which relies on the mitochondria-restricted expression of a fluorescent reporter that is sensitive to pH and hence changes excitation wavelength within lysosomes (mt-mKeima) – can be adapted to a variety of human and mouse cancer cell lines and/or straightforwardly implemented on fluorescence microscopy platforms.