Methods for Detection of Autophagy in Mammalian Cells. 2019

Bindu Singh, and Sangeeta Bhaskar
Product Development Cell, National Institute of Immunology, New Delhi, India.

Autophagy is a conserved catabolic process that degrades cytoplasmic constituents in the lysosome and thus contributes to the maintenance of intracellular homeostasis. The process of autophagy has been involved in many physiological and pathological processes. Therefore, there is a developing need to identify, quantify, and manipulate the autophagic process accurately in the cells. As autophagy involves dynamic and complex processes, therefore various approaches are needed to study this process precisely. In this chapter, we have tried to elaborate the approaches and methods to monitor autophagy, with a primary focus on mammalian macroautophagy. Autophagy induction can be detected using Western blotting of LC3 (marker protein for autophagosomes) in which LC3-II levels represent the quantity of autophagosomes formed on induction to a particular stimulus. This can also be confirmed by puncta formation assay using confocal microscopy. Further, the autophagic flux can be examined using bafilomycin A1 as inhibitor of autophagosome-lysosome fusion and acidification of lysosomal compartments, thereby leading to accumulation of autophagosomes which is represented by high LC3-II levels. The autophagolysosomal degradation or proteolysis which is the last step of autophagy can be analyzed by DQ-BSA assay.

UI MeSH Term Description Entries
D008247 Lysosomes A class of morphologically heterogeneous cytoplasmic particles in animal and plant tissues characterized by their content of hydrolytic enzymes and the structure-linked latency of these enzymes. The intracellular functions of lysosomes depend on their lytic potential. The single unit membrane of the lysosome acts as a barrier between the enzymes enclosed in the lysosome and the external substrate. The activity of the enzymes contained in lysosomes is limited or nil unless the vesicle in which they are enclosed is ruptured or undergoes MEMBRANE FUSION. (From Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed). Autolysosome,Autolysosomes,Lysosome
D005453 Fluorescence The property of emitting radiation while being irradiated. The radiation emitted is usually of longer wavelength than that incident or absorbed, e.g., a substance can be irradiated with invisible radiation and emit visible light. X-ray fluorescence is used in diagnosis.
D000067996 RAW 264.7 Cells A transformed macrophage cell line isolated from ASCITES of mice infected with ABELSON MURINE LEUKEMIA VIRUS. RAW 264.7 Cell Line,264.7 Cell, RAW,264.7 Cells, RAW,Cell, RAW 264.7,Cells, RAW 264.7,RAW 264.7 Cell
D000071182 Autophagosomes Large spherical double-layered structures which function in AUTOPHAGY to engulf intracellular components such as ORGANELLES or pathogens. Their outer membrane then fuses with the LYSOSOME (forming the AUTOLYSOSOME) and the inner membrane and contents are digested by lysosomal HYDROLASES. Phagophores,Autophagosome,Phagophore
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001343 Autophagy The segregation and degradation of various cytoplasmic constituents via engulfment by MULTIVESICULAR BODIES; VACUOLES; or AUTOPHAGOSOMES and their digestion by LYSOSOMES. It plays an important role in BIOLOGICAL METAMORPHOSIS and in the removal of bone by OSTEOCLASTS. Defective autophagy is associated with various diseases, including NEURODEGENERATIVE DISEASES and cancer. Autophagocytosis,ER-Phagy,Lipophagy,Nucleophagy,Reticulophagy,Ribophagy,Autophagy, Cellular,Cellular Autophagy,ER Phagy
D051379 Mice The common name for the genus Mus. Mice, House,Mus,Mus musculus,Mice, Laboratory,Mouse,Mouse, House,Mouse, Laboratory,Mouse, Swiss,Mus domesticus,Mus musculus domesticus,Swiss Mice,House Mice,House Mouse,Laboratory Mice,Laboratory Mouse,Mice, Swiss,Swiss Mouse,domesticus, Mus musculus
D059748 Proteolysis Cleavage of proteins into smaller peptides or amino acids either by PROTEASES or non-enzymatically (e.g., Hydrolysis). It does not include Protein Processing, Post-Translational. Protein Degradation,Protein Digestion,Degradation, Protein,Degradations, Protein,Digestion, Protein,Digestions, Protein,Protein Degradations,Protein Digestions,Proteolyses
D018613 Microscopy, Confocal A light microscopic technique in which only a small spot is illuminated and observed at a time. An image is constructed through point-by-point scanning of the field in this manner. Light sources may be conventional or laser, and fluorescence or transmitted observations are possible. Confocal Microscopy,Confocal Microscopy, Scanning Laser,Laser Microscopy,Laser Scanning Confocal Microscopy,Laser Scanning Microscopy,Microscopy, Confocal, Laser Scanning,Confocal Laser Scanning Microscopy,Confocal Microscopies,Laser Microscopies,Laser Scanning Microscopies,Microscopies, Confocal,Microscopies, Laser,Microscopies, Laser Scanning,Microscopy, Laser,Microscopy, Laser Scanning,Scanning Microscopies, Laser,Scanning Microscopy, Laser

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