Many researchers, especially those new to the field, need to determine which criteria are essential for demonstrating autophagy, either for the purposes of their own research, or in the capacity of a manuscript or grant review. 1 Acceptable standards are an important issue, particularly considering that each of us may have his/her own opinion regarding the answer. Unfortunately, the answer is in part a “moving target” as the field evolves. 2 This can be extremely frustrating for researchers who may think they have met those criteria, only to find out that the reviewers of their papers have different ideas. Conversely, as a reviewer, it is tiresome to raise the same objections repeatedly, wondering why researchers have not fulfilled some of the basic requirements for establishing the occurrence of an autophagic process. In addition, drugs that potentially modulate autophagy are increasingly being used in clinical trials, and screens are being carried out for new drugs that can modulate autophagy for therapeutic purposes. Clearly it is important to determine whether these drugs are truly affecting autophagy, and which step (s) of the process is affected, based on a set of accepted criteria. Accordingly, we describe here a basic set of contemporary guidelines that can be used by researchers to plan and interpret their experiments, by clinicians to evaluate the literature with regard to autophagy-modulating therapies, and by both authors and reviewers to justify or criticize an experimental approach.

Several fundamental points must be kept in mind as we establish guidelines for the selection of appropriate methods to monitor autophagy. 2 Importantly, there are no absolute criteria for determining autophagic status that are applicable in every biological or experimental context. This is because some assays are inappropriate, problematic or may not work at all in particular cells, tissues or organisms. 3-6 For example, autophagic responses to drugs may be different in transformed versus nontransformed cells, and in confluent versus nonconfluent cells, or in cells grown with or without glucose. 4 In addition, these guidelines are likely to evolve as new methodologies are developed and current assays are superseded. Nonetheless, it is useful to establish guidelines for acceptable assays that can reliably monitor autophagy in many experimental systems. It is important to note that in this set of guidelines the term “autophagy” generally refers to macroautophagy; other autophagy-related processes are specifically designated when appropriate. For the purposes of this review, the autophagic compartments (Fig. 1) are referred to as the sequestering (pre-autophagosomal) phagophore (PG; previously called the isolation or sequestration membrane 5, 6), 7 the autophagosome (AP), 8 the amphisome (AM; generated by the fusion of autophagosomes with endosomes), 9 the lysosome, the autolysosome (AL; generated by fusion of autophagosomes or amphisomes with a lysosome), and the autophagic body (AB; generated by fusion and release of the internal autophagosomal compartment into the vacuole in fungi and plants). Except for cases of highly stimulated autophagic sequestration (Fig. 2), autophagic bodies are