Table 5.

Methods to measure autophagy in vivo ^1,2.

Method	Advantages	Limitations
Transgenic mice expressing GFP-MAP1LC3 and fluorescence microscopy	Allow the formation of autophagosomes to be studied. Used to study macroautophagy and mitophagy.	Do not permit the formation of autolysosome to be studied as GFP loses its fluorescence at acidic pH in lysosomes. Other transgenic mice are required [88] No measurement of autophagic flux. Cells possess auto-fluorescent punctate structures such as lipofuscin that is detectable in the green spectra. Always compare to non-transgenic control littermates [66].
mCherry-GFP-MAP1LC3 and mRFP-GFP-MAP1LC3 mouse and fluorescence microscopy	High time resolution.	Technical difficulty in distinguishing RFP/GFP double-positive and single positive punctae. Lack of performance to measure the basal autophagic flux.
GFP-MAP1LC3-RFP-LC3∆G mRNA (injected in animal eggs) and fluorescence microscopy	Measure the basal (low) and induced autophagy flux in embryos and tissues of zebrafish and mice [70].

¹ SBI-0206965 (Adooq Bioscience), a potent and selective inhibitor of ULK1, can be used to inhibit autophagy in vivo; is given intraperitoneally into mice at 2 mg/kg body weight in DMSO, once per day for 7 days [89]; other autophagy blockers can be used in vivo as control, e.g., CQ/HCQ, NH₄Cl, bafilomycin A1 (in certain strict conditions), colchicine, vinblastine, and the inhibitor of lysosomal enzymes leupeptide [66]. ² Measurement of chaperone-mediated autophagy (CMA) activity in vivo has been described [90]. See abbreviations in the abbreviations section.