Monitoring Autophagy in Live Cells Without Transfection

Transfection is the process of introducing nucleic acids into cells. In cell-based assays, transfection is typically used to investigate protein expression and gene function by introducing nucleic acids (transiently or permanently) into the cell and studying how the cell’s internal machinery modulates gene expression in response. Transfection is most commonly achieved by one of three methods: chemical-based (calcium phosphate, polymers (DEAE-dextran, cationic), activated dendrimer, etc.), cationic lipid-mediated (lipofection) and/or physical (electroporation, cell squeezing, direct microinjection, magnetofection etc.). However, not all methods can be applied to all cell types and experimental circumstances. Transfection efficiencies are dependent upon multiple factors such as the cell type, health of the cells, culture methods being applied, quality and quantity of the DNA being introduced, and the experimental demands. Transfection by way of either chemical-based, lipid-based, or physical methods present a variety of limitations that can affect transfection efficiencies.
Popular chemical-based transfection means such as calcium phosphate co-precipitation are prone to variability; small pH changes can compromise the efficiency of calcium phosphate transfection into the cell and size and quality of the precipitate is critical for successful gene transfer and expression to occur. Likewise, chemical methods used to introduce DNA using DEAE-dextran can become difficult because endocytotic mechanisms that uptake these reagents can accumulate in toxic quantities in the cell. The result is often varying transfection efficiencies which have been found to produce as low as 10% or less successful gene transfer in primary cells. Transfection by way of lipid-mediated methods demonstrates a disadvantage by not having the flexibility to be applied to all cell types unless they have the appropriate frequency of liposomes which is variable across cell types and based on cell health. Lastly, physical methods such as microinjection and electroporation impose different obstacles that both influence transfection efficiency. Microinjections are technically demanding, laborious, and time-consuming. Electroporation does not always lead to high transfection efficiencies especially if studying cells in suboptimal conditions to understand the pathological activities at the molecular level of a disease state.

Autophagy is a dynamic, multi-step stress-induced protective mechanism that can be regulated at several steps. Autophagic activity is typically low under basal conditions, but can be markedly up-regulated by a variety of physiological stimuli such as nutrient starvation, hypoxia, energy depletion, endoplasmic reticulum stress, elevated temperature, high density growth conditions, hormonal stimulation, pharmacological agent treatment, innate immune signaling, and in diseases such as viral, bacterial or parasitic infections as well as various protein aggregopathies (e.g., Alzheimer’s, Huntington’s and Parkinson’s disease), heart disease and acute pancreatitis. The role of autophagic activity in the pathology of cancer, infectious diseases, neurodegeneration, cardiovascular disease, and diabetes has become widely recognized and commonly studied. A reduction in autophagic function is also considered a characteristic of the aging process.

Suboptimal conditions may be imposed on a cell population to study autophagy in a cell-based assay. Cells are subjected to hostile conditions such as nutrient depletion and chemical or environmental stress to induce lysosomal-mediated bulk clearance of cellular debris that autophagic processes utilize. Using transfection-based methods to study such a process in a cell-based assay can often lead to lengthy optimizations and transfection efficiency validation to ensure results are accurate.

To avoid these time consuming, laborious adjustments and validations, Enzo offers a transfection-free CYTO-ID Autophagy Detection Kit and CYTO-ID Autophagy Detection Kit 2.0 for monitoring autophagy in live cells. The kits measure autophagic vacuoles and monitor autophagic flux in lysosomally inhibited live cells using a novel dye that selectively labels accumulated autophagic vacuoles. The dye has been optimized through the identification of titratable functional moieties and allows for minimal staining of lysosomes while exhibiting a bright fluorescent signal once incorporated into pre-autophagosomes, autophagosomes and autolysosomes. Ultimately, this provides the opportunity to study autophagy with a no-transfection quantitative assay which can be utilized on live cells across microscopy, flow cytometry, and microplate platforms. The CYTO-ID Autophagy Detection Kit 2.0 provides a brighter, more photostable dye, which binds to the same acidic, autophagic compartments and can facilitate high-throughput screening of activators and inhibitors of autophagy.

Key Features

• No transfection required. Eliminates the need for time and effort-consuming transfection efficiency validation required with LC3-fusion protein transfection.
• Brighter, more photostable edition of our proprietary dye with titratable moieties specific for selectively staining autophagic vesicles.
• Selective and comprehensive staining that allows for the measurement and differentiation between autophagic flux and autophagolysosome accumulation.
• Rapidly quantifies autophagy in native heterogeneous cell populations.
• Validated under a wide range of conditions and with small molecular modulations known to influence autophagy pathways.
• Optimized protocols for microplate assays, fluorescence microscopy and flow cytometry.
• Negligible staining of lysosomes reduces background seen with other dyes.
• Facilitates high-throughput screening of activator and inhibitors of autophagy.

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