Strategies for imaging mitophagy in high-resolution and high-throughput.

We describe two approaches for measuring mitophagy in mammalian cells using stable cells expressing EGFP-LC3 - Mito-DsRed to mark early phase of mitophagy and Mitochondria-EGFP - LAMP1-RFP stable cells for late events of mitophagy. Both the assays showed good spatial and temporal resolution in wide-field, confocal and super-resolution microscopy with high-throughput adaptable capability. A limited compound screening allowed us to identify a few new mitophagy inducers. Compared to the current mitophagy tools, mito-Keima or mito-QC, the assay described here determines the direct delivery of mitochondrial components to the lysosome in real time mode with accurate quantification if monoclonal cells expressing a homogenous level of both probes are established. Since the assay described here employs real-time imaging approach in a high-throughput mode, the platform can be used both for siRNA screening or compound screening to identify key regulators of mitophagy at decisive stages. PMID: 29092745 [PubMed - as supplied by publisher]
Source: European Journal of Cell Biology - Category: Cytology Authors: Tags: Eur J Cell Biol Source Type: research