Kim Luu1 , Wini Luty2
1IntelliCyt Corporation, Albuquerque, New Mexico, USA
2Enzo Life Sciences, Farmingdale, New York, USA
Featured Product: CYTO-ID® Autophagy Detection Kit
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Autophagy has rapidly become an important target in drug discovery. However, current methods to assay autophagy-related responses are not easily adapted to high throughput screening campaigns. This application note demonstrates the use of Enzo’s Cyto-ID® Autophagy Dye and IntelliCyt’s MultiCytCell Membrane Integrity Dye and iQue® Screener as a platform to detect changes to basal autophagy in cell-based assays. The autophagy response is profiled in a screening compatible workflow for 14 known effectors of autophagy across 6 different cell lines in a mix-and-read, no wash assay.
Introduction
Autophagy (in particular, macroautophagy) is a pathway that delivers a portion of a cell’s cytoplasm to the lysosome for degradation. Turnover of proteins and long-lived organelles by autophagy are a normal part of the homeostatic activities of a cell. During periods of metabolic stress, such as nutrient limitation, autophagy is upregulated to generate the necessary macromolecules for new synthesis. In addition to its importance in the stress response, autophagy is known to play a role in neurodegenerative, liver, muscle, and cardiac diseases, as well as cancer, aging, and clearance of infections(1). The involvement of autophagy in a wide variety of disorders has made it an attractive target in drug discovery.
The current understanding of the molecular mechanisms of autophagy are well-reviewed in the literature(2). In brief, autophagy initiates with the formation of a phagophore, which is a membrane that begins to surround a portion of the cytoplasm (Figure 1). The phagophore develops into a fully enclosed vesicle called an autophagosome that completely sequesters a portion of the cytoplasm. The autophagosome then docks and fuses with a lysosome, creating an autolysosome, and the contents of the vesicle and the vesicle itself are degraded.
Most methods of autophagy detection use quantification or visualization of autophagy-related intracellular markers. A commonly used marker is LC3-II because it is known to uniquely associate with the fully formed autophagosome(3). Microscopy or western blotting can be used to quantify LC3-II as a measure of autophagosome accumulation or depletion in response to a treatment. Engineered cell lines are often used, in which case an LC3/fluorescent protein fusion is overexpressed within the cell. These methods require complex assay development, can be cumbersome in high throughput screenings, and in the case of engineered cell lines, raise questions of physiological disease relevance.
In this application note, the ability of the iQue® Screener to detect changes to basal autophagy is characterized in multiple cell lines. CYTO-ID® Autophagy Dye and IntelliCyt’s MultiCyt Cell Membrane Integrity reagent are used to quantify autophagy and cell viability from the same samples. Fourteen known modulators of autophagy, selected because they span multiple mechanisms of action, are shown to cause changes in autophagy dye staining that varies by cell type. Notably some compounds cause significant cell toxicity in addition to perturbations in autophagy. The membrane integrity endpoint measurement is utilized as a primary quality control “flag” whereby samples with less than 80% viability are not evaluated for autophagy as the biological relevance at that point is ambiguous.
The assay platform offers unique advantages for high throughput screening: it is homogeneous, requiring only addition of the dye to your assay and 30 minute incubation. Importantly, this assay allows screening your cellular model of choice without having to engineer a specific cell line. The ability to multiplex and simultaneously detect cell viability in addition to autophagy provides a powerful mechanism for uncovering compound toxicity that could contribute to artifacts in a potential autophagy screen.
