A No-transfection, Quantitative Assay for Monitoring Autophagy in Live Cells

• No transfection required
• Proprietary dye includes titratable moieties specific for selectively staining autophagic vesicles
• Protocol validated with known inhibitors and activators of autophagic activity
• Rapidly quantifies autophagy in native heterogeneous cell populations
• Eliminates need for time and effort-consuming transfection efficiency validation required with LC3-GFP transfection
• Selective and comprehensive staining, allows measurement and differentiation between autophagic flux and autophagolysosome accumulation
• Negligible staining of lysosomes reduces background seen with other dyes
• Facilitates high-throughput screening of activators and inhibitors of autophagy

Cyto-ID® Autophagy Detection Kit measures autophagic vacuoles and monitors autophagic flux in live cells using a novel dye that selectively labels autophagic vacuoles. The dye has been optimized through the identification of titratable functional moieties that allow for minimal staining of lysosomes while exhibiting bright fluorescence upon incorporation into pre-autophagosomes, autophagosomes, and autolysosomes (autophagolysosomes). The assay offers a rapid and quantitative approach to monitoring autophagy in live cells without the need for cell transfection.

Mechanism of Action
The probe is a cationic amphiphilic tracer (CAT) dye that rapidly partitions into cells in a similar manner as drugs that induce phospholipidosis.
Careful selection of titratable functional moieties on the dye prevents its accumulation within lysosomes, but enables labeling of vacuoles associated with the autophagy pathway.