Real-Time Autophagic Flux Measurements in Live Cells Using a Novel Fluorescent Marker DAPRed
Arnold Sipos,
Kwang- Kim,
Juan Alvarez,
Edward Crandall
Affiliations
Arnold Sipos
Will Rogers Institute Pulmonary Research Center and Hastings Center for Pulmonary Research, Keck School of Medicine, University of Southern California, Los Angeles, CA, USADepartment of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
Kwang- Kim
Will Rogers Institute Pulmonary Research Center and Hastings Center for Pulmonary Research, Keck School of Medicine, University of Southern California, Los Angeles, CA, USADepartment of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA, Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA, Department of Pharmacology and Pharmaceutical Sciences, Alfred E. Mann School of Pharmacy and Pharmaceutica
Juan Alvarez
Will Rogers Institute Pulmonary Research Center and Hastings Center for Pulmonary Research, Keck School of Medicine, University of Southern California, Los Angeles, CA, USADepartment of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
Edward Crandall
Will Rogers Institute Pulmonary Research Center and Hastings Center for Pulmonary Research, Keck School of Medicine, University of Southern California, Los Angeles, CA, USADepartment of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA, Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, CA, USA
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.
