Table 1.

Methods used to monitor autophagy in adult neurogenesis studies

Assay	In vitro or	Readout	Caveats of technique	Reference
	In vivo		(from Klionsky DJ et al., 2016)
Transmission Electron Microscopy (TEM)	In vitro	Vacuoles containing cytoplasmic contents (autophagosomes or autolysosomes)	Difficult to quantify using unbiased method. Variability in cell areas sampled.	[55]
Detection of LC3-I and LC3-II	In vitro	Increased band intensity of LC3-II and increased ratio of LC3-II over LC3-I	Difficult to distinguish between upregulation of autophagosome formation or blockage of autophagic degradation without using lysosomal inhibitors.	[55]
				[56]
				[57]
				[58]
Detection of LC3-II and p62 in presence of chloroquine	In vitro	Lower LC3-II and higher p62 accumulation	Changes in p62 levels can be specific to cell-type and context being studied.	[47]
				[50]
p62 sequestosome/degradation assay	In vitro	Accumulation of p62 in presence of proteasome inhibitor	Not always a correlation between increase in autophagy and decrease in P62 levels.	[57, 58]
LC3 immunofluorescence	In vitro	Reduced in number of LC3 puncta	Not all LC3 puncta represent LC3-II and correspond to autophagosomes.	[50]
GFP-LC3 puncta	In vitro	Increase in number of GFP-LC3 puncta	High levels of GFP-LC3 can associate with ubiquitinated aggregates to lead to misinterpretation about the number of autophagic structures. Reflects only a snapshot of the numbers of autophagy-related structures.	[56]
GFP-LC3 transgenic mice and detection of LC3-II in presence of chloroquine	In vitro and in vivo	Phenotype cells expressing GFP-LC3 puncta. Lower LC3-II accumulation	GFP-LC3 expression driven by CAG promoter, thus intensity of the GFP signal may represent CAG promoter activity, not autophagic activity.	[49]
mRFP-GFP-LC3 tandem construct	In vitro	Increases in percentage of mRFP-only positive LC3 puncta.	Colocalization of GFP and mRFP might be seen in the case of impaired proteolytic degradation within autolysosomes, or altered lysosomal pH	[58]
mCherry-EGFP-LC3 retrovirus	In vivo	mCherry+ puncta (autolysosomes) around cell body.	Difficult to distinguish between reduced autophagosome production or enhanced autolysosomal degradation without using lysosomal inhibitors.	[40]