Table 2.

MAP1LC31-based standard assay for assessing autophagy and their limitations ¹.

Techniques	Special Attention, Limitations and Pitfalls
Western blot assay (Figure 2)	Examining the expression of MAP1LC3-I and -II in the presence and absence of protease inhibitors is an absolute requirement 2. Certain autophagosome-lysosome fusion competitors inhibit MTORC1, which initiates the induction of autophagy process [56,61,62]. The choice of inhibitors is decisive. For example, CQ can activate MAP1LC3-II formation independently from autophagy [63]. Specific detection of MAP1LC3-II is dependent on the type of antibody used. The majority of commercialized antibodies cross-react with several MAP1LC3 isoforms [64,65]. Antibodies may show different affinity for MAP1LC3-I and -II, and together with differing protein stability of the non- and lipidated forms western blotting bands require careful interpretation.
Fluorescence microscopy for detecting endogenous MAP1LC3	Discrimination between immatured, not yet closed and mature autophagosomes is required as both appear as punctate. Characterization of MAP1LC3 puncta. Advanced image analysis software’s (e.g., Top Hat algorithm of MetaMorph version 7.0 by Molecular Devices and G-Count by G-Angstrom) is a very useful tool to measure MAP1LC3 puncta [56]. Quantification can also be made manually by a trained and blinded observer. Discrimination of true autophagosomes devoid of MAP1LC3 aggregates, which are formed due to the aggregate prone proteins and autophagy-independent manner can be difficult.
Fluorescence microscopy for detecting reporters (e.g., GFP-MAP1LC3, mRFP-GFP-MAP1LC3, …)	Tissues from GFP-MAP1LC3 transgenic mice expresses more auto-fluorescence punctate structures [66]. Lack of GFP-MAP1LC3 expression in GFP-MAP1LC3 transgenic mice brain was observed, unlike other tissues. Cells deficient of ATG proteins, especially ATG5, would not generate MAP1LC3 punctate structures [67]. However, not all MAP1LC3 punctate structures are indicative of autophagy [58]. Loss of time-dependent fluorescence (GFP-MAP1LC3) intensity, but not mutant MAP1LC3, was observed [68]. In GFP- or mRFP-GFP-MAP1LC3 constructs, labelling may not give absolute results, especially if the pH of lysosomes is altered in pathological situations (as in lupus, for example, in which the mean lysosomal pH is raised [35]). Use of samples with or without inhibitors should be maintained for the better comparison (except for a few probes, e.g., GFP-MAP1LC3-RFP-MAP1LC3∆G). In terms of GFP-MAP1LC3-RFP-LC3∆G probe, more time (>2 h) is needed to observe significant changes in fluorescence ratio. Clone selection (transfection studies) should be monitored [69,70]. Assays based on the red fluorescent protein Keima cannot be used with fixed cells because the assay completely relies on lysosomal acidity [71].
Flow cytometry	Detects the different forms of endogenous MAP1LC3 (incl. MAP1LC3-I, MAP1LC3-II) proteins without any discrimination. Improved speed and statistical power when determining autophagic flux using tandem MAP1LC3 fusion proteins. Requires isolation of subcellular vesicles (e.g., autophagosomes, lysosomes) to highlight possible defects in the expression of endogenous MAP1LC3 protein levels [72]. Necessity to handle cell samples immediately [73].
Multispectral imaging flow cytometry	Combines features of flow cytometry with the imaging content of fluoresecent microscopy [74,75] Allows for detection of MAP1LC3 dot formation representative for MAP1LC3-II. Visualization of MAP1LC3 co-localization with lysosomal markers or other proteins.
Bioluminescence	Using a luminescent peptide to tag endo- and exogenous MAP1LC3 [76]. Allows easy detection and sensitive quantification of specific MAP1LC3 isoforms. Adapted to perform high throughput screening of compounds, for example. Small marker peptide allows for facilitated endogenous gene tagging using CRISPR/Cas9 technology. Does not allow detection of MAP1LC3 punctae formation.
MAP1LC3B time-resolved fluorescence transfer (TR-FRET) assay	Homogenous, mix-and-read assay that takes advantage of the required proximity of the donor and acceptor species for the generation of signal [77].
Electron microscopy (Figure 3)	Difficulty in discriminating the various types of vesicles (autolysosomes, endosomes, amphisomes, lysosomes) Difficulty to evaluate autophagy dynamics. No direct information obtained on lysosomal degradation. Time consuming. Technical errors, e.g., poor-fixation, sometimes leads to over or under looking observations [78]. Conventional methods, but not advanced electron microscopy methods, are not suitable to determine the volume and size of the inner cell compartments, due to the thin sections [60].
Long-lived protein degradation	Proteasome inhibitors should be used to specify the action of autophagy. Labelling efficiency is always a question, e.g., special culture media, without methionine, is required in non-radioactive labelling [79].
LDH sequestration assay	Lysosomal inhibitors always need to be used to measure autophagic flux [80].
Dextran sequestration assay	Loading fluorescently labelled dextran into cells is a delicate operation [81,82].

¹ A few other conventional methods and/or nonspecific methods, such as isotope release from long-lived protein degradation (radiolabeling long-lived proteins with radioactive amino acid residues, such as [¹⁴C]-leucine, [³H]-leucine, [¹⁴C]-valine, or [³⁵S]-methionine) and LDH assay (using LDH as a cargo) are not reviewed here as they are not routinely used. ²We are well aware that determining the ATG protein levels or the number of autophagosomes alone does not provide the overall estimation of autophagic activity since the process is very dynamic, and MAP1LC3-II can quickly degrade within the lysosomes. In fact, the two opposite scenarios, namely induction of autophagy or blockade in the downstream steps of autophagy leading to defective degradation, can result in an increased number of autophagosomes. Therefore, it is necessary to evaluate the autophagic flux by measuring the level of expression of MAP1LC3-I and -II in the presence of lysosomal inhibitors such as bafilomycin A1 or others. See abbreviations in the abbreviations section.