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The precise chemical structure of the dye is proprietary. The probe is a cationic amphiphilic tracer (CAT) dye that rapidly partitions into cells in a way similar to many cationic drugs. The dye is taken up by passive diffusion across the plasma membrane bilayer and does not require protein binding or transporter activity. Careful selection of titratable functional moieties on the dye prevents its accumulation within lysosomes, but enables labeling of vacuoles associated with the autophagy pathway. Additionally, an enhancement in the fluorescence emission intensity of the dye likely occurs upon compartmentalization with the lamellar membrane structures associated with autophagic vesicles. We and others have generated data documenting the dye's selectivity for autophagic vesicles.
Treatments that induce autophagolysosome accumulation, such as chloroquine or baflimyocin treatment, lead to a significant increase in fluorescence signal (250-300%). Rapamycin induces autophagic response that can be blocked by 3-MA treatment. Amino acid starvation in Earl's balanced salt solution (EBSS) leads to the accumulation of dye in autophagic vesicles after as little as 1 hour and this accumulation is reversed by re-feeding the cells. We have successfully detected autophagy vacuoles using a host of pharmacological modulators acting on different components of the pathway (phagophore through autophagolysosome). Many of these agents are included in the Screen-Well® Autophagy library.