An optimized procedure for quantitative analysis of mitophagy with the mtKeima system using flow cytometry

Mitophagy is the process by which mitochondria are selectively targeted and removed via autophagic machinery to maintain mitochondrial homeostasis in the cell. Recently, flow cytometry-based assays that utilize the fluorescent mtKeima reporter system have allowed for quantitative assessment of mitophagy at a single-cell level. However, clear guidelines for appropriate flow cytometry workflow and downstream analysis are lacking and studies using flow cytometry in mtKeima-expressing cells often display incorrect and arbitrary binary mitophagic or nonmitophagic cutoffs that prevent proper quantitative analyses. In this paper we propose a novel method of mtKeima data analysis that preserves subtle differences present within flow cytometry data in a manner that ensures reproducibility. In this report we propose an outline for the transfection of mtKeima into a cell monolayer, cell preparation and downstream analysis using flow cytometry and associated software, with emphasis on a novel data processing technique.