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A Fluorescence Microplate-Based Assay Workflow for High-Throughput Screening of Compounds Modulating Autophagy in Living Cells下載
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February 02, 2011
Authors: Paul Held, Peter Banks, BioTek Instruments, Inc., Winooski, Vermont; Dee Shen, Wayne Patton, Enzo Life Sciences, Farmingdale, New York
Autophagy is a normal degradative pathway that involves the sequestration of entire organelles, protein complexes, and misfolded proteins in a membrane vacuole called the autophagosome. The autophagosomal vacuole is subsequently delivered to the lysosome where it is degraded into its essential constituents and recycled back to the cytoplasm. Autophagy plays important roles in diverse biological events, including responses to nutrient limitation, tumor suppression, the immune response, and aspects of neurodegeneration. However, experimental methods to monitor this process in mammalian cells are limited and often highly qualitative. For example, LC3, a mammalian homologue of the ubiquitin-like (UBL) protein Atg8, has been used as a specific marker of autophagosomes in mammals. However, current methods to quantify autophagic activity using LC3 are time-consuming and labor-intensive, involving the physical counting of fluorescent punctate signal forming within the cells. We have developed and validated a novel cell-based autophagy assay using a 488 nm-excitable green-emitting fluorescent probe to highlight the various vacuolar components of the autophagy pathway. We demonstrated that the accumulation of autophagy probe was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Furthermore, a population of this dye-labeled vesicle co-localizes with LC3. We have validated this fluorescent probe under a wide range of conditions known to modulate autophagy pathways. We have optimized the use of the EL406™ Combination Washer Dispenser to automatically aspirate media, wash cells and dispense reagents for the assay, allowing, for the first time, easy quantitation of autophagy using a convenient fluorescence microplate-based HTS format. This assay enables kinetic analysis of the autophagy pathway and is able to distinguish between increases in autophagic flux vs. autophagic vacuole accumulation.