In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

• Keywords
• Autophagosome, LC3, cancer, flux, lysosome, macroautophagy, neurodegeneration, phagophore, stress, vacuole,
• MeSH
• Autophagy * physiology   MeSH
• Autophagosomes MeSH
• Biomarkers MeSH
• Biological Assay standards   MeSH
• Humans MeSH
• Lysosomes MeSH
• Autophagy-Related Proteins metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Guideline MeSH
• Names of Substances
• Biomarkers MeSH
• Autophagy-Related Proteins MeSH

Physiological adjustments accompanying insect cold acclimation prior to cold stress have been relatively well explored. In contrast, recovery from cold stress received much less attention. Here we report on recovery of drosophilid fly larvae (Chymomyza costata) from three different levels of cold stress: supercooling to -10 °C, freezing at -30 °C, and cryopreservation at -196 °C. Analysis of larval CO2 production suggested that recovery from all three cold stresses requires access to additional energy reserves to support cold-injury repair processes. Metabolomic profiling (targeting 41 metabolites using mass spectrometry) and custom microarray analysis (targeting 1,124 candidate mRNA sequences) indicated that additional energy was needed to: clear by-products of anaerobic metabolism, deal with oxidative stress, re-fold partially denatured proteins, and remove damaged proteins, complexes and/or organelles. Metabolomic and transcriptomic recovery profiles were closely similar in supercooled and frozen larvae, most of which successfully repaired the cold injury and metamorphosed into adults. In contrast, the majority of cryopreseved larvae failed to proceed in ontogenesis, showed specific metabolic perturbations suggesting impaired mitochondrial function, and failed to up-regulate a set of 116 specific genes potentially linked to repair of cold injury.

• MeSH
• Drosophilidae * genetics   metabolism   MeSH
• Stress, Physiological * MeSH
• Cryopreservation * methods   MeSH
• Larva MeSH
• Metabolomics methods   MeSH
• Preservation, Biological MeSH
• Cold-Shock Response MeSH
• Gene Expression Profiling MeSH
• Freezing * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• Keywords
• LC3, autolysosome, autophagosome, chaperone-mediated autophagy, flux, lysosome, macroautophagy, phagophore, stress, vacuole,
• MeSH
• Autophagy * physiology   MeSH
• Biological Assay methods   standards   MeSH
• Humans MeSH
• Computer Simulation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, N.I.H., Extramural MeSH
• Guideline MeSH