Autophagy is a conserved homeostatic mechanism involved in cellular homeostasis
and many disease processes. Although it was first described in yeast cells
undergoing starvation, we have learned over the years that autophagy gets
activated in many stress conditions and during development and aging in
mammalian cells. Understanding the fundamental mechanisms underlying autophagy
effects can bring us closer to better insights into the pathogenesis of many
disease conditions (e.g., cardiac muscle necrosis, Alzheimer’s disease,
and chronic lung injury). Due to the complex and dynamic nature of the
autophagic processes, many different techniques (e.g., western blotting,
fluorescent labeling, and genetic modifications of key autophagy proteins) have
been developed to delineate autophagy effects. Although these methods are valid,
they are not well suited for the assessment of time-dependent autophagy
kinetics. Here, we describe a novel approach: the use of DAPRed for autophagic
flux measurement via live cell imaging, utilizing A549 cells, that can visualize
and quantify autophagic flux in real time in single live cells. This approach is
relatively straightforward in comparison to other experimental procedures and
should be applicable to any in vitro cell/tissue models.
Key features
• Allows real-time qualitative imaging of autophagic flux at single-cell level.
• Primary cells and cell lines can also be utilized with this technique.
• Use of confocal microscopy allows visualization of autophagy without disturbing
cellular functions.