Table 2.

Methods monitoring mitophagy in liver disease.

Methods	Pros	Cons	Applications in Liver and Liver Disease Study
Electron Microscopy (EM)	Provides mitochondria-containing autophagosome and autolysosome ultrastructure	Limitations in quantification, steady-state rather than detecting flux	ALD [113,114,115,119] DILI [155,159] I/R [168] HBV/HCV [189]
Immunoelectron Microscopy (IEM)	Provides mitochondria-containing autophagosome and autolysosome ultrastructure and related proteins	Not quantitative	ALD [113,119] HBV/HCV [189]
Co-localization of LC3 with a Mitochondrial Protein	Large number of cells	The fluorescence-labeled LC3 aggregates may be misleading Not objective and robust Will not be able to detect LC3-independent mitophagy, microautophagy or MDVs	ALD [119,122] NAFLD [86,133,134,136,137,138,139] DILI [158] I/R [166] HBV/HCV [178,179,189] Cancer [217,218]
Autophagy/Mitophagy Marker Proteins	Objective Quantitative	Non-specific The intracellular distribution of marker proteins is more important than its total amount, total amount does not equal activity, only steady state	ALD [114,115,119,120,122] NAFLD [133,134,136,137,138,139,140] I/R [165,167,168,169,172,173] HBV/HCV [178,179,189,190,192] Cancer [216,217,218]
Mitochondrial Mass	Objective Quantitative	Only reflect steady state, rather than flux, nor the degradation or the initiation process of mitophagy. Mitochondrial outer membrane proteins are also degraded by proteasome.	ALD [114,116,119,122] NAFLD [134,140] DILI [155,157,159] HBV/HCV [192] Cancer [216]
pH-Sensitive Fluorescent Probe	Specific High image quality Apply in vivo and in vitro	The expression level of fluorescent proteins varies in different cells/tissues Not robust and easy to be dependent on individuals who are performing the quantification. Also, the half-life of the red puncta in the lysosomes may be dependent on cellular context and conditions, e.g., the activities of the lysosomal proteases	NAFLD [86] DILI [159] Cancer [216]