Measurement of Bulk Autophagy by a Cargo Sequestration Assay
The nonselective bulk sequestration of cytoplasm and its subsequent delivery to lysosomes for degradation was originally defined as autophagy or macroautophagy. However, both terms are now increasingly being applied in a generic sense to encompass the many recently described mechanisms for selective sequestration and degradation of individual cellular elements. We will therefore use the term bulk autophagy to denote the non-exclusive and largely nonselective process.
Bulk autophagy can be measured directly by a cargo sequestration assay, using a cargo marker representative of total cytoplasm. The assay described here measures the sequestration and accumulation of the ubiquitous cytosolic protein lactate dehydrogenase (LDH) in the sedimentable autophagic vacuoles of cells incubated with an inhibitor of intravacuolar degradation such as bafilomycin or leupeptin. Electrodisruption of the plasma membrane followed by centrifugal sedimentation of the “cell corpses” (which retain their organelles in an intact state, bound to the cytoskeleton) is a convenient, efficient, and reproducible way to separate the small fraction of sequestered, sedimentable LDH from the major pool of cytosolic LDH.
Key wordsAutophagy Bulk autophagy Bafilomycin Cargo assay Cell corpse Cell culture Electrodisruption Lactate dehydrogenase Lysosome Phagophore Sequestration
This work has been generously supported by grants from the Norwegian Cancer Society, the Research Council of Norway, the University of Oslo, the Nansen Foundation, the Blix Family Fund, and Astri and Birger Torsted's Legacy.
Disclosure: No conflict of interest was disclosed.