title: Autophagic flux determination in vivo and ex vivo.
abstract: Autophagy is a highly dynamic process that mediates the degradation of cellular constituents inside lysosomes. It is characterized by the formation of autophagosomes, double membrane organelles that engulf cytosolic components and organelles and degrade their contents upon fusion with lysosomes. Upregulation of autophagy in response to specific stimuli can be determined by evaluating autophagic flux. This is achieved by comparing the number of autophagosomes in the absence and presence of lysosomal inhibitors. While the determination of autophagic flux in isolated cells is well-documented, few studies have described its determination in tissues or in vivo. Here, we describe the evaluation of autophagic flux both in vivo and ex vivo in several tissues, after treatment with lysosomal inhibitors and exposure to classical autophagy-inducing stimuli. This method uses LC3 lipidation, as determined by Western blot, fluorescence microscopy and flow cytometry. Our findings demonstrate that autophagic flux can be evaluated in vivo and ex vivo in several tissues.
authors: Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain. Electronic address: lesteban@cib.csic.es.
authors: Lorena
authors: L
authors: Esteban-Martínez
authors: Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain. Electronic address: pboya@cib.csic.es.
authors: Patricia
authors: P
authors: Boya
mesh_terms: D000818:Animals; D001343:Autophagy / Q000235:genetics*; D002531:Cerebellum / Q000378:metabolism / Q000648:ultrastructure; D007976:Leupeptins / Q000737:chemistry; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure*; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D012160:Retina / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D007976:Leupeptins; C032854:leupeptin
keywords: Autophagic flux; Cerebellum; Ex vivo autophagy determination; GFP-LC3 mice; In vivo autophagy determination; Liver; Retina