In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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A MP-activated protein kinase (AMPK) is a phylogenetically conserved heterotrimer protein consisting of three subunits, ␣, ␤, and ␥, each of which has at least two isoforms (1-3). AMPK is activated by physiological stimuli such as exercise and by hormones, including adiponectin and leptin, as well as by pathological stresses such as glucose deprivation, hypoxia, oxidant stress, and osmotic shock (1-3). Increases in the ratio of AMP to ATP activate AMPK by a number of mechanisms, including direct allosteric activation and covalent modification due to activation by an AMPK kinase (1-3), which phosphorylates the ␣-subunit on threonine-172 (Thr172) (4,5). Once activated, AMPK phosphorylates multiple targets both in vivo and in vitro (1-3), including several biosynthetic enzymes such as acetyl-CoA carboxylase, hydroxymethylglutaryl-CoA reductase, glycogen synthase, and both neuronal and endothelial nitric oxide (NO) synthase (eNOS) (2,6).NO, a free radical gaseous molecule synthesized by the action of the enzyme eNOS, is the most important factor in maintaining vascular homeostasis (7). Endothelium-derived NO promotes vasodilation and inhibits platelet aggregation, leukocyte adherence, and vascular smooth muscle proliferation (7). It has been reported that regulation of eNOS activity is regulated by reversible phosphorylation (8,9) and its interaction with other proteins, such as heat shock protein (hsp)-90 (10,11). For example, AMPK has been demonstrated to phosphorylate eNOS at serine-1179 (Ser1179; equal to human Ser1179) with concomitant activation of eNOS in ischemic heart (7) and in intact human aortic endothelial cells activated with oxidants (peroxynitrite [ONOO Ϫ ]) (12), 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (13), and adiponectin (14,15). However, whether AMPK regulates eNOS in vivo remains to be determined.Metformin, a bioguanide derivative (dimethylbiguanide), is one of the most commonly used drugs for the treatment of type 2 diabetes (16,17). Metformin improves insulin sensitivity, decreases insulin levels, and reduces elevated plasma glucose (16,17). In addition to its insulinsensitizing effects, metformin has also been shown to have direct vascular effects (18 -22). Most importantly, met- AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; AMPK, 5Ј-AMPactivated kinase; AMPK-CA, constitutively active AMPK kinase; BAEC, bovine aortic endothelial cell; cGMP, cyclic GMP; eNOS, endothelial nitric oxide synthase; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein; hsp, heat shock protein; ICAM, intracellular adhesion molecule; L-NAME, L-nitro-arginine methyl ester; 3-NT, 3-nitrotyrosine; Ser1179, serine-1179; SOD, superoxide dismutase; Thr172, threonine-172; PDK, phosphoinositide-dependent kinase; VCAM, vascular cell adhesion molecule.