Methods for mitochondrial health assessment by High Content Imaging System

Mitochondria are important organelles responsible for energy production. Mitochondrial dysfunction relates to various pathological diseases. The investigation of mitochondrial heath is critical to evaluate the cellular status. Herein, we demonstrated an approach for determining the status of mitochondrial health by observing mitochondrial H₂O₂ (one type of ROS), membrane potential, and morphology (fragmentation and length) in live primary fibroblast cells. The cells were co-stained with fluorescent dyes (Hoechst 33342 and MITO-ID® Red/MitoPY1/JC-10) and continuously processed by the High Content Imaging System. We employed the Operetta CLS^TM to take fluorescent images with its given quickness and high resolution. The CellProfiler image analysis software was further used to identify cell and mitochondrial phenotypes in the thousand fluorescent images.•We could quantitatively analyze fluorescent images with high-throughput and high-speed detection to track the alteration of mitochondrial status.•The MMP assay is sensitive to FCCP even at the concentration of 0.01 µM.•The fibroblast cells treated with stress inducers (H₂O₂, FCCP, and phenanthroline) revealed a significant change in mitochondrial health parameters, with more ROS accumulation, depolarized MMP, increased fragmentation, and reduced length of mitochondria.