A novel quantitative flow cytometry-based assay for autophagy.
Autophagy is a cellular degradation process with an increasingly recognised importance in many biological pathways such as nutrient sensing, stress responses and development. We present a straightforward assay for autophagy which combines the sensitivity of the EGFP-LC3 reporter protein with the throughput capacity and quantitative power of flow cytometry. Because saponin extraction is specific for the non-autophagosome associated EGFP-LC3-I form of the protein, flow cytometry can be used to measure total fluorescence of saponin extracted HOS-EGFP-LC3 cells as a measure of the levels of autophagosome associated EGFP-LC3-II. Combined with inhibitors of degradation, we have adapted this assay to differentiate between constitutive and induced autophagy and to quantify the changes in flux of the system. Moreover, using direct antibody staining for the endogenous LC3 protein, we have extended this assay to the detection of autophagosome formation in non-transfected cells.
Eng
Kai Er
KE
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
Panas
Marc D
MD
Karlsson Hedestam
Gunilla B
GB
McInerney
Gerald M
GM
D000818:Animals; D001343:Autophagy / Q000187:drug effects; D001681:Biological Assay / Q000379:methods*; D002738:Chloroquine / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D012503:Saponins / Q000378:metabolism
C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D012503:Saponins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002738:Chloroquine