Technical Routes and Result Interpretation in Cell Autophagy Detection

Cell autophagy is an important homeostatic regulatory process in which cells degrade cytoplasmic proteins, damaged organelles, protein aggregates, and some exogenous components through the lysosomal system. The core of autophagy detection is not merely observing autophagosome number or changes in a single marker protein, but determining whether autophagic flux is complete, namely whether autophagosome formation, maturation, fusion with lysosomes, and substrate degradation proceed continuously.

Keywords: cell autophagy; autophagic flux; LC3; p62/SQSTM1; Beclin 1; ATG proteins; autophagosome; autolysosome; lysosomal function; Western blot; fluorescence imaging; transmission electron microscopy; mitophagy

1、Basic Principles of Cell Autophagy Detection

1.1 Detection Nodes in the Autophagy Process

(1) Autophagy initiation

Autophagy initiation is usually associated with nutrient deprivation, energy stress, oxidative stress, endoplasmic reticulum stress, drug stimulation, or organelle damage. The ULK1 complex, Beclin 1, VPS34 complex, and ATG proteins participate in autophagic membrane nucleation and elongation. Detection at this stage may include Beclin 1, ATG5, ATG7, ULK1 phosphorylation, and changes in mTOR/AMPK signaling. However, these indicators can only suggest the regulatory status of autophagy and cannot independently prove enhanced autophagic flux.

(2) Autophagosome formation

During autophagosome formation, LC3-I undergoes lipidation to form membrane-bound LC3-II, which localizes to the autophagosomal membrane. Increased LC3-II, increased LC3 puncta, or elevated autophagosome numbers may all indicate increased autophagosome formation. However, an increase in autophagosomes may result either from enhanced autophagy induction or from impaired autophagosome degradation, so flux experiments are required for interpretation.

(3) Autophagosome maturation and lysosomal fusion

Mature autophagosomes fuse with lysosomes to form autolysosomes, where substrates are degraded in an acidic environment by lysosomal hydrolases. If lysosomal acidification, membrane fusion, or protease activity is impaired, LC3-II and p62 may accumulate. In this case, the apparent increase in autophagy-related structures may actually reflect blockade at the degradation stage of autophagy.

(4) Substrate degradation

Selective autophagy receptors such as p62/SQSTM1 and NBR1 bind ubiquitinated substrates and interact with LC3, after which they enter lysosomes for degradation during autophagy. A decrease in p62 usually suggests enhanced autophagic substrate degradation, whereas p62 accumulation suggests insufficient substrate clearance or autophagy blockade. However, p62 is also regulated by transcription, oxidative stress, and inflammatory signaling, and should not be used as the only criterion.

1.2 Core Logic of Autophagy Detection

(1) Distinguishing autophagosome number from autophagic flux

Increased LC3-II, increased autophagosomes, and increased LC3 fluorescent puncta are all autophagy-related phenotypes, but they do not directly equal enhanced autophagy. Autophagic flux emphasizes the dynamic process and requires comparison of LC3-II, p62, and autophagy reporter signals before and after lysosomal blockade.

(2) Combining structural, protein, and functional indicators

Reliable autophagy detection generally requires at least three levels of evidence: LC3-II or LC3 puncta to reflect autophagosome formation, p62/NBR1 to reflect substrate degradation, and Bafilomycin A1, chloroquine, or dual-fluorescence LC3 systems to reflect flux status. A single indicator is suitable only as suggestive evidence.

(3) Simultaneously evaluating cellular status

Cell death, protein synthesis inhibition, lysosomal damage, mitochondrial depolarization, and cell cycle changes can all affect autophagy detection results. Autophagy experiments should simultaneously evaluate cell viability, apoptosis/necrosis, lysosomal acidification, total protein degradation, and treatment-dose toxicity.

Table 1 Core Indicators and Interpretation Boundaries in Cell Autophagy Detection

Detection level	Common indicators	What it can indicate	Interpretation limitations
Autophagy initiation	ULK1, Beclin 1, VPS34, ATG proteins	Whether autophagy regulatory pathways are activated or inhibited	Cannot directly indicate flux changes
Autophagosome formation	LC3-II, LC3 puncta, autophagosome number	Changes in autophagosome formation or LC3 lipidation	Cannot distinguish increased formation from impaired degradation
Autophagic substrate degradation	p62/SQSTM1, NBR1, ubiquitinated proteins	Whether substrates accumulate or are cleared	Affected by transcription, protein synthesis, and proteasome activity
Autophagic flux	LC3-II changes before and after lysosomal inhibitors	Dynamic status of autophagosome formation and degradation	Requires optimization of inhibitor concentration and treatment time
Lysosomal function	LAMP1, LAMP2, LysoTracker, cathepsin activity	Lysosomal acidification, fusion, and degradation capacity	Cannot independently represent autophagy initiation
Ultrastructure	Autophagosomes and autolysosomes under TEM	Presence of autophagy-related membranous structures	Limited ability to determine flux

2、LC3-Related Detection Methods

2.1 LC3-I/LC3-II Western Blot

(1) Detection principle

LC3 is the most commonly used marker protein in cell autophagy detection. Cytosolic LC3-I is lipidated to form LC3-II, which localizes to the autophagosomal membrane. Western blot usually detects the conversion from LC3-I to LC3-II and uses LC3-II relative to an internal control or the LC3-II/LC3-I ratio as a reference indicator for autophagosome formation.

(2) Result interpretation

Increased LC3-II suggests an increase in autophagy-related membrane structures, but the reason may be enhanced autophagy induction or impaired autophagosome-lysosome fusion or degradation. If LC3-II further accumulates after adding Bafilomycin A1 or chloroquine, this indicates that autophagosomes are still being continuously formed and entering the degradation pathway. If LC3-II does not increase further after blockade, insufficient autophagosome generation or pre-existing flux obstruction should be considered.

(3) Technical points

LC3-II has a small molecular weight and is sensitive to gel concentration, transfer efficiency, and antibody specificity. Lysis conditions must sufficiently extract membrane-bound LC3-II to avoid signal underestimation caused by incomplete lysis. Basal LC3 levels differ greatly among cell types, so absolute grayscale values should not be directly compared across cell lines.

2.2 LC3 Immunofluorescence and Puncta Analysis

(1) Detection principle

Under basal conditions, LC3 usually shows diffuse distribution. When autophagosome formation increases, LC3 may form punctate aggregates. LC3 immunofluorescence or a GFP-LC3 expression system can be used to observe the number, area, and spatial distribution of intracellular LC3 puncta.

(2) Result interpretation

Increased LC3 puncta suggest an increase in autophagosomes or LC3-positive membrane structures, but this alone cannot prove enhanced autophagic flux. If increased LC3 puncta are accompanied by p62 reduction and lysosomal inhibitors can further increase LC3 puncta, enhanced autophagic flux is more strongly supported. If increased LC3 puncta are accompanied by p62 accumulation, blockade at the autophagic degradation stage is more likely.

(3) Technical points

GFP-LC3 overexpression may form nonspecific protein aggregates and generate false-positive puncta. Transfection amount and expression level should be controlled, and empty vector, positive induction, lysosomal blockade, and untreated controls should be included. Image quantification should calculate puncta number per cell, puncta area, proportion of positive cells, or fluorescence intensity distribution rather than relying only on representative images.

2.3 Main Limitations of LC3 Detection

(1) Not flux-specific

Increased LC3-II and LC3 puncta cannot independently distinguish enhanced autophagy induction from degradation blockade.

(2) Interference from noncanonical LC3-related processes

LC3 may participate in LC3-associated phagocytosis, endosomal membrane modification, and certain noncanonical membrane processes. In immune cells, phagocytes, infection models, and membrane-trafficking abnormality models, LC3-positive structures do not necessarily represent classical autophagosomes.

(3) Strong time dependence

Autophagy is a dynamic process. At different time points, LC3-II may first increase and then decrease, while p62 may first accumulate and then degrade. A time-course design is therefore preferable to testing a single endpoint.

Table 2 Comparison of LC3-Related Detection Methods

Method	Detection object	Main advantage	Main limitation	Recommended use
LC3 Western blot	LC3-I, LC3-II	Relatively clear quantification, suitable for flux experiments	Requires interpretation with lysosomal blockade	Autophagosome formation and flux analysis
LC3 immunofluorescence	LC3 puncta	Allows observation of spatial distribution	Cannot independently determine flux	Autophagosome localization and intracellular distribution
GFP-LC3 reporter system	LC3-positive membrane structures	Suitable for imaging and dynamic observation	Overexpression may cause aggregation	Observation of autophagosome formation
LC3 immunohistochemistry	LC3 expression in tissue sections	Preserves tissue localization	Limited quantification and flux interpretation	Autophagy-related localization in tissue samples
LC3 and LAMP1 colocalization	Relationship between autophagosomes and lysosomes	May indicate fusion-stage changes	Colocalization does not equal completed degradation	Autophagosome maturation analysis

3、p62/SQSTM1 and Autophagic Substrate Detection

3.1 Detection of p62 Protein Level

(1) Detection principle

p62/SQSTM1 is a selective autophagy receptor that binds ubiquitinated substrates and interacts with LC3 through its LC3-interacting region. It is subsequently delivered to lysosomes for degradation during autophagy. Therefore, p62 protein level is often used to evaluate autophagic substrate clearance.

(2) Result interpretation

Increased LC3-II accompanied by decreased p62 usually supports enhanced autophagic flux. Increased LC3-II accompanied by increased p62 suggests autophagic substrate accumulation or impaired lysosomal degradation. If p62 decreases but LC3-II shows no obvious change, p62 transcriptional suppression, reduced protein synthesis, or other degradation pathways should be considered.

(3) Technical points

p62 is regulated by Nrf2, NF-κB, oxidative stress, inflammatory stimulation, and proteostasis stress. It is recommended to detect both p62 protein and SQSTM1 mRNA to distinguish transcriptional regulation from autophagy-mediated protein degradation.

3.2 Detection of Selective Autophagy Substrates

(1) NBR1

NBR1 is similar to p62 and can serve as an auxiliary indicator of selective autophagy substrates. NBR1 reduction may support enhanced substrate degradation, but it still requires interpretation together with LC3 and flux experiments.

(2) Ubiquitinated protein aggregates

When autophagy is blocked, ubiquitinated proteins and insoluble protein aggregates may accumulate. Protein quality control status can be evaluated by Western blot of ubiquitinated proteins, immunofluorescence, or insoluble protein fraction analysis.

(3) Specific organelle substrates

Selective autophagy should be evaluated around the target organelle. Mitophagy can be assessed using TOM20, COX IV, VDAC, PINK1, and Parkin. ER-phagy can be assessed using ER-phagy receptors and ER marker proteins. Lipophagy requires integration of lipid droplet staining, LC3 colocalization, and lipid metabolism indicators.

Table 3 Indicators for Autophagic Substrate Detection

Indicator	Common methods	Meaning of change	Interpretation notes
p62/SQSTM1	Western blot, immunofluorescence, immunohistochemistry	Decrease suggests enhanced substrate degradation; increase suggests substrate accumulation or blockade	Regulated by transcription and stress signaling
NBR1	Western blot, immunofluorescence	Assists evaluation of selective autophagy substrate degradation	Should not be used alone as a flux indicator
Ubiquitinated proteins	Western blot, immunofluorescence	Accumulation suggests protein degradation stress or autophagy blockade	Proteasome activity also affects results
TOM20/COX IV/VDAC	Western blot, immunofluorescence	Reduction may suggest mitochondrial clearance	Must be combined with mitochondrial mass and cell viability
Insoluble protein aggregates	Fraction extraction, immunofluorescence	Aggregate accumulation suggests proteostasis abnormality	Need to distinguish autophagy from proteasome effects

4、Autophagic Flux Detection Methods

4.1 Lysosomal Inhibitor Method

(1) Detection principle

The core of autophagic flux detection is comparing LC3-II or autophagic substrates before and after lysosomal inhibitor treatment. Common reagents include Bafilomycin A1, chloroquine, hydroxychloroquine, NH4Cl, and lysosomal protease inhibitors. If LC3-II further accumulates after lysosomal degradation is blocked, this indicates that autophagosomes continue to form and enter the degradation process.

(2) Result interpretation

If LC3-II increases in the treatment group and increases further after adding a lysosomal inhibitor, enhanced autophagic flux is supported. If LC3-II is already elevated but the inhibitor does not further increase it, blockade at the autophagic degradation stage should be considered. p62 usually accumulates in the presence of lysosomal inhibitors; if it does not accumulate, treatment time, protein synthesis, and detection sensitivity should be checked.

(3) Technical points

Inhibitor concentration and treatment time should be determined by pilot experiments. Bafilomycin A1 inhibits lysosomal acidification and may affect autophagosome-lysosome fusion-related processes. Chloroquine raises lysosomal pH but may also affect endocytosis, membrane trafficking, and cell toxicity. Flux experiments should avoid prolonged treatment that causes secondary cell injury.

4.2 mRFP-GFP-LC3 Dual-Fluorescence System

(1) Detection principle

mRFP-GFP-LC3 uses the fact that GFP is sensitive to acidic environments whereas RFP is relatively acid-resistant to distinguish autophagosomes from autolysosomes. In neutral autophagosomes, both GFP and RFP signals are present, appearing as yellow puncta. After entry into acidic autolysosomes, the GFP signal is quenched and mainly red puncta remain.

(2) Result interpretation

An increase in yellow puncta suggests increased autophagosomes. An increase in red puncta suggests autophagosomes have entered acidic lysosomes and formed autolysosomes. If yellow puncta increase markedly but red puncta do not, autophagosome-lysosome fusion impairment or abnormal lysosomal acidification should be considered.

(3) Technical points

Expression level of the reporter protein should be controlled to avoid nonspecific aggregation caused by overexpression. Imaging parameters should be kept consistent, and starvation induction, lysosomal blockade, and untreated controls should be included. Quantification should separately count yellow puncta, red puncta, and the red/yellow puncta ratio.

4.3 GFP-LC3-RFP-LC3ΔG System

(1) Detection principle

This system uses the feature that GFP-LC3 can be degraded by autophagy, whereas RFP-LC3ΔG does not participate in autophagic degradation. The GFP/RFP ratio is used to reflect autophagic flux. When autophagy is enhanced, GFP-LC3 degradation increases and the GFP/RFP ratio decreases.

(2) Application characteristics

This method is suitable for quantitative assessment of autophagic flux changes and can reduce the subjectivity of simple puncta counting. It is suitable for drug screening, genetic intervention, and dynamic flux analysis.

(3) Notes

Stable expression of the reporter system must be ensured, and the effects of treatment on GFP/RFP protein expression, translation efficiency, fluorescence stability, and cell viability must be excluded.

Table 4 Comparison of Autophagic Flux Detection Methods

Method	Detection logic	Main advantage	Main limitation	Application scenario
LC3-II plus lysosomal inhibitor	Compare LC3-II accumulation before and after blockade	Direct flux interpretation, suitable for Western blot	Inhibitors may cause cytotoxicity and nonspecific effects	Routine autophagic flux detection
p62 plus lysosomal inhibitor	Compare whether substrate degradation is blocked	Observes substrate clearance status	p62 is affected by transcriptional regulation	Auxiliary judgment of substrate degradation
mRFP-GFP-LC3	Distinguishes autophagosomes from autolysosomes	Visualizes flux process	Depends on transfection and imaging quality	Live-cell or fixed-cell imaging
GFP-LC3-RFP-LC3ΔG	Uses fluorescence ratio to reflect flux	Suitable for quantification and screening	Requires stable reporter system	Drug screening, mechanism studies
LC3 and LAMP1 colocalization	Assesses relationship between autophagosomes and lysosomes	Provides relatively clear spatial information	Colocalization does not equal completed degradation	Fusion-stage analysis

5、Lysosomal Function and Fluorescent Dye Detection

5.1 LysoTracker and LysoSensor

(1) Detection principle

LysoTracker and LysoSensor label acidic organelles and are used to observe lysosomal acidification and acidic compartment changes. Autophagy ultimately depends on lysosomal degradation, so abnormal lysosomal acidification directly affects autophagic flux.

(2) Result interpretation

Enhanced acidic lysosomal signal may indicate increased lysosome number or acidification, but may also result from lysosomal accumulation, damage, or autophagy blockade. If LC3-II elevation is accompanied by abnormally enhanced LysoTracker signal and p62 accumulation, blockade at the autophagic degradation stage should be considered.

(3) Notes

LysoTracker is not an autophagy-specific dye and can only serve as a lysosomal status indicator. Dye concentration, incubation time, cell density, and imaging parameters all affect signal intensity.

5.2 MDC and Cyto-ID

(1) MDC staining

MDC can label autophagic vacuole-like structures, but its specificity is limited and it is easily affected by other membranous structures. MDC alone cannot provide sufficient evidence for autophagy occurrence or enhanced flux.

(2) Cyto-ID staining

Cyto-ID can be used to detect autophagy-related vesicles and is commonly used for flow cytometry or fluorescence microscopy. Its advantage is operational simplicity and suitability for screening, but verification with LC3, p62, or flux experiments is still required.

(3) Interpretation principle

Fluorescent dye-based methods are suitable for rapid screening and auxiliary observation. They should not replace LC3-II, p62, and autophagic flux detection. If dye signal increases but classical autophagy indicators do not support the conclusion, interpretation should be cautious.

5.3 Lysosomal Proteins and Enzyme Activity Detection

(1) Cathepsin activity

Activities of lysosomal proteases such as Cathepsin B, Cathepsin D, and Cathepsin L reflect degradation capacity. If autophagosome formation is normal but cathepsin activity decreases, autophagic substrates may still fail to degrade efficiently.

(2) LAMP1/LAMP2

LAMP1 and LAMP2 are lysosome-associated membrane proteins and can be used to observe lysosome number, distribution, and colocalization with LC3-positive structures. LAMP2 abnormalities may affect autophagosome-lysosome fusion and substrate degradation.

(3) Lysosomal pH

Lysosomal acidification is essential for autophagic degradation. Drug treatment, genetic intervention, or cell damage that raises lysosomal pH may cause p62 accumulation, LC3-II elevation, and impaired substrate clearance.

Table 5 Lysosome- and Autophagy-Related Fluorescent Detection Methods

Method	Detection object	Relationship with autophagy	Limitation
LysoTracker	Acidic lysosomal compartments	Reflects lysosome number and acidification status	Not autophagy-specific
LysoSensor	Changes in lysosomal pH	Determines whether lysosomal acidification is abnormal	Affected by dye conditions and imaging parameters
MDC	Autophagic vacuole-like structures	Can assist observation of autophagy-related vesicles	Low specificity
Cyto-ID	Autophagy-related vesicles	Suitable for screening and flow cytometry	Requires validation with classical indicators
Cathepsin activity assay	Lysosomal protease function	Assesses substrate degradation capacity	Does not reflect autophagy initiation
LAMP1/LAMP2 immunofluorescence	Lysosomal membrane structures	Can analyze autophagosome-lysosome relationship	Colocalization does not equal completed degradation

6、Electron Microscopy Detection

6.1 Detection Value

(1) Ultrastructural observation

Transmission electron microscopy can directly observe double-membrane autophagosomes, single-membrane autolysosomes, membranous structures engulfing cytoplasmic contents, and vesicle morphology at different degradation stages. It is an important method for confirming autophagy-related structures.

(2) Structural resolution advantage

Electron microscopy can observe organelle damage, mitochondrial engulfment, membranous structure formation, and degradation contents inside lysosomes. It has high value for mitophagy, organelle-selective autophagy, and ultrastructural analysis of tissue samples.

6.2 Interpretation Points

(1) Autophagosomes

Typical autophagosomes are double-membrane structures containing undegraded cytoplasmic components or organelles. Single-membrane vacuoles or ordinary lysosomes alone should not be directly defined as autophagosomes.

(2) Autolysosomes

Autolysosomes are mostly single-membrane structures containing degrading cytoplasmic components with heterogeneous electron density. Their presence suggests that autophagosomes have entered the degradation stage, but still does not provide quantitative flux information alone.

(3) Quantification methods

The number of autophagosomes/autolysosomes per cell, number of autophagic structures per unit area, or proportion of autophagic vesicles containing mitochondria can be counted. Sample fixation, sampling region, section quality, and field selection significantly affect results.

6.3 Limitations

Electron microscopy requires demanding sample preparation, provides limited flux information, and only covers limited observation fields. It should be used as a structural validation method rather than the sole basis for autophagy detection. If autophagosome accumulation is observed by electron microscopy, LC3-II, p62, and lysosomal blockade experiments are still needed to evaluate autophagic flux.

Table 6 Interpretation of Autophagic Structures Under Electron Microscopy

Structure type	Morphological features	Biological meaning	Interpretation notes
Autophagosome	Double membrane, engulfing cytoplasm or organelles	Autophagosome formation	Must be distinguished from ordinary vacuoles
Autolysosome	Single membrane, contains degradation material	Autophagosome has entered degradation stage	Cannot independently quantify flux
Mitochondria-containing structure	Mitochondrion surrounded by membranous structure	May suggest mitophagy	Should be combined with mitochondrial markers
Vacuole-like structure	Irregular morphology, few contents	May represent damage or nonspecific vacuoles	Should not be directly defined as autophagy
Increased lysosomes	High electron density or complex contents	Active or impaired degradation system	Requires lysosomal function testing

7、Molecular-Level and Signaling Pathway Detection

7.1 Autophagy-Related Proteins

(1) ATG proteins

ATG5, ATG7, ATG12, ATG16L1, and other proteins participate in autophagosome formation. Their expression changes or gene knockdown can be used to verify autophagy dependence. If a treatment increases LC3-II and this change disappears after ATG5 or ATG7 knockdown, the process is more likely to depend on the classical autophagy pathway.

(2) Beclin 1

Beclin 1 participates in autophagy initiation and membrane nucleation. Changes in Beclin 1 may suggest the regulatory status of autophagy initiation, but cannot independently represent changes in autophagic flux.

(3) ULK1 and mTOR/AMPK signaling

mTOR inhibition, AMPK activation, and changes in ULK1 phosphorylation may indicate regulation of autophagy by nutrient and energy-sensing pathways. Starvation, rapamycin, Torin1, and energy stress often affect autophagy through these pathways. Signaling pathway indicators should be interpreted together with LC3, p62, and flux experiments.

7.2 mRNA Detection

(1) Applicable scope

qPCR can detect changes in MAP1LC3, BECN1, ATG5, ATG7, SQSTM1, and other gene expression levels, and is used to analyze the transcriptional regulatory background of autophagy.

(2) Limitations

Autophagy is primarily a dynamic degradation process, and mRNA expression cannot directly reflect autophagic flux. Especially when SQSTM1 mRNA is upregulated, p62 protein may not decrease significantly even if autophagic degradation is enhanced.

7.3 Genetic Validation

(1) Gene knockdown or knockout

siRNA, shRNA, CRISPR, and other approaches can be used to knock down ATG5, ATG7, BECN1, and ULK1 to verify whether a phenotype depends on autophagy.

(2) Functional rescue

If autophagy phenotypes and cellular functional changes are restored by re-expressing the relevant gene in a knockdown background, the credibility of the mechanistic conclusion is strengthened.

(3) Selective autophagy validation

For mitophagy, ER-phagy, lipophagy, and other forms of selective autophagy, specific receptors, organelle markers, and colocalization analysis must be combined. Total LC3 changes alone are insufficient.

Table 7 Molecular-Level Detection Indicators

Detection category	Common indicators	What it can indicate	Main limitation
Autophagy initiation	ULK1, Beclin 1, VPS34	Whether initiation signals are regulated	Cannot directly indicate flux
Autophagosome formation	ATG5, ATG7, ATG12, ATG16L1	Whether autophagy machinery participates	Expression changes do not equal functional changes
mTOR/AMPK signaling	p-mTOR, p-AMPK, p-ULK1	Nutrient and energy signaling regulation	Pathways are complex and require functional experiments
Transcriptional detection	MAP1LC3, SQSTM1, BECN1 mRNA	Transcriptional regulatory background	Cannot replace protein-level flux detection
Genetic validation	ATG5/ATG7 knockdown or knockout	Autophagy dependence	May cause long-term adaptive changes

8、Product Selection for Cell Autophagy Detection

Table 8 Autophagy Induction and Inhibition Reagents

Cat. No.	Product Name	Grade/Specification	Applicable detection stage	Application positioning
E743125	Earle's Balanced Salt Solution (with Ca2+xa0& Mg2+)		Starvation/nutrient deprivation induction	Used to establish nutrient stress or autophagy induction conditions; can serve as a basic treatment system for autophagy induction experiments
S129300	STF-62247	≥98%	Autophagy induction	Used for pharmacological autophagy induction, suitable for combination with LC3-II, p62, and flux assays
Lib412034	Autophagy Compound Library		Autophagy regulation screening	Used for screening autophagy inducers, inhibitors, or regulatory compounds
R408790	Rapamycin (AY-22989)	Moligand™, 10mM in DMSO	Autophagy induction control	Used for mTOR-dependent autophagy induction; commonly used as a positive control
S115842	Rapamycin	Moligand™, ≥98%(HPLC)	Autophagy induction control	Used as a positive inducer in Western blot, fluorescence imaging, and flux assays
S293790	Rapamycin	Moligand™, ≥95%(HPLC)	Autophagy induction	Used in mTOR inhibition-related autophagy studies
T420960	Torin 1	Moligand™, 2mM in DMSO	Autophagy induction	Used for studies of autophagy activation related to mTORC1/2 inhibition
D110019	D-(+)-Trehalose Anhydrous	≥99%	mTOR-independent autophagy induction	Used for mTOR-independent autophagy induction studies
D427220	D-(+)-Trehalose Anhydrous	10mM in DMSO	Autophagy induction	Used for comparison of autophagy induction conditions
M486160	3-Methyladenine	autophagy inhibitor	Autophagy initiation/nucleation blockade	Used for PI3K-related autophagy initiation inhibition and autophagy-dependence validation
M424419	3-Methyladenine	10mM in DMSO	Autophagy initiation/nucleation blockade	Used for pharmacological blockade of autophagosome formation
M129496	3-Methyladenine	≥98%	Autophagy initiation/nucleation blockade	Used as an early autophagy inhibition control
W409260	Wortmannin (KY 12420)	Moligand™, 10mM in DMSO	Autophagy initiation inhibition	Used for intervention in VPS34/PI3K-related autophagy initiation stages
W100984	Wortmannin	Moligand™, ≥98%	Autophagy initiation inhibition	Used to validate blockade of the autophagosome formation stage
A1423681	ATG7-IN-1	≥99%	Autophagosome formation intervention	Used for inhibition of ATG7-dependent autophagy processes
A1497260	ATG7-IN-2	Moligand™, 10 mM in DMSO	Autophagosome formation intervention	Used for pharmacological inhibition experiments involving ATG7
A1424123	ATG7-IN-2	≥99%	Autophagosome formation intervention	Used for ATG7-dependent mechanism validation
A1423893	ATG7-IN-3	≥99%	Autophagosome formation intervention	Used for ATG7-related autophagy inhibition studies
B1423685	Beclin1-ATG14L interaction inhibitor 1	≥99%	Autophagy initiation/membrane nucleation intervention	Used for studies of autophagy mechanisms related to Beclin1-ATG14L interaction
B1423730	Beclin1-Bcl-2 interaction inhibitor 1		Autophagy initiation regulation	Used for studies of Beclin1-Bcl-2 axis-related autophagy regulation
T648510	Tat-beclin 1	≥99%	Autophagy induction	Used for Beclin 1-related autophagy induction studies
T414076	Tat-beclin 1 acetate(Tat-BECN1 acetate)	≥98%	Autophagy induction	Used to induce autophagy and validate Beclin 1-related pathways
T1422310	Tat-beclin 1 scrambled	≥99%	Autophagy induction control	Used as a sequence control in Tat-beclin 1-related experiments
T1422555	Tat-beclin 1 scrambled TFA	≥98%	Autophagy induction control	Used to exclude nonspecific effects of Tat-beclin 1

Table 9 Autophagy Staining, Lysosomal Function, and Fluorescence Imaging Reagents

Cat. No.	Product Name	Grade/Specification	Applicable detection stage	Application positioning
C1520245	Autophagy/Cytotoxicity Dual-Color Detection Kit (MDC/PI Method)	BioReagent,Biological Stain,for microscopy,sterile,for fluorescence analysis	Autophagic vacuole-like structures/cytotoxicity	Used to label autophagy-related vesicles with MDC and evaluate cytotoxicity with PI
C1511655	Autophagy Staining and Detection Kit (MDC Method)	BioReagent,Biological Stain,for microscopy	Autophagic vacuole-like structures	Used for rapid fluorescence observation of autophagy-related vesicles; requires LC3/p62 validation
C1520234	Autophagy Green Detection Kit (MDC Method)	BioReagent,Biological Stain,for microscopy,sterile,for fluorescence analysis	Autophagic vacuole-like structures	Used for green fluorescence detection of autophagy-related vesicles; suitable for microscopy and screening assays
L1455325	LysoTracker Blue DND-22		Lysosomal acidification/localization	Used for acidic lysosomal compartment labeling and auxiliary assessment of the autophagic degradation stage
L647096	LysoTracker Red	≥97%	Lysosomal acidification/localization	Used for lysosome localization and colocalization analysis with LC3 and p62
L1455226	LysoTracker Yellow HCK 123	≥98%	Lysosomal acidification/localization	Used to observe acidic lysosomal compartments
L1455145	LysoSensor PDMPO	≥98%	Lysosomal acidification function	Used to evaluate lysosomal pH changes and autophagic degradation conditions
O1501802	Hoechst 33342 Staining Solution	Suitable for Immunofluorescence(IF), BioReagent, ready-to-use, Biological Stain, for fluorescence analysis, Biological dye grade, for microscopy, for cell culture, 1.0 mg/ml in H₂O	Fluorescence imaging auxiliary localization	Used for nuclear localization and cell counting in LC3, p62, LAMP1, and other immunofluorescence assays
B422759	bisBenzimide H 33342 trihydrochloride	10mM in DMSO	Fluorescence imaging auxiliary localization	Used for nuclear staining of live or fixed cells
B113845	bisBenzimide H 33342 trihydrochloride	≥98%	Fluorescence imaging auxiliary localization	Used for cell nuclear localization in autophagy fluorescence detection
B755401	Bisbenzimide H 33342 Fluorochrome, Trihydrochloride	Cell-permeable, adenine-thymine-specific fluorescent stain.	Fluorescence imaging auxiliary localization	Used for nuclear staining and image normalization
D489987	4',6-Diamidino-2-phenylindole Dihydrochloride	≥97%	Fluorescence imaging auxiliary localization	Used for nuclear localization in immunofluorescence
D106471	4',6-Diamidino-2-phenylindole Dihydrochloride	≥98%	Fluorescence imaging auxiliary localization	Used for nuclear counterstaining in LC3/p62/LAMP1 colocalization imaging
D423005	4',6-Diamidino-2-phenylindole Dihydrochloride	10mM in DMSO	Fluorescence imaging auxiliary localization	Used for nuclear staining in autophagy-related immunofluorescence experiments
D598342	DAPI staining solution (ready to use)		Fluorescence imaging auxiliary localization	Used for nuclear localization and cell counting in autophagy fluorescence imaging

Table 10 LC3- and p62-Related Detection Tools

Cat. No.	Product Name	Grade/Specification	Applicable detection stage	Application positioning
A743522	AAV-mCherry-GFP-LC3B (AAV9)	10^13vp/ml	Autophagic flux	Used to distinguish autophagosomes from autolysosomes; suitable for flux observation in cells or animal models
A743648	Ad-GFP-LC3B		Autophagosome formation	Used to observe GFP-LC3B puncta and autophagosome formation
A743656	Ad-mCherry-GFP-LC3B		Autophagic flux	Used to distinguish autophagosomes and autolysosomes by yellow/red puncta
A743666	AdPlus-mCherry-GFP-LC3B		Autophagic flux	Used for imaging autophagosome maturation and lysosomal fusion
L747631	Lenti-EGFP-LC3B	10^9TU/ml	Autophagosome formation	Used to establish stable LC3B reporter cell lines
L747634	Lenti-mCherry-EGFP-LC3B	10^9TU/ml	Autophagic flux	Used for autophagic flux observation in stable expression systems
P748882	pCMV-GFP-LC3B		Autophagosome formation	Used for transient expression of GFP-LC3B and observation of LC3 puncta
P748885	pCMV-mCherry-GFP-LC3B		Autophagic flux	Used to distinguish autophagosomes from autolysosomes
Ab156564	LC3B Mouse mAb	Carrier Free,ExactAb™,Azide Free,Validated,High Performance,PBS Only,≥95%(SDS-PAGE),1.0 mg/mL	LC3 Western blot/immunofluorescence/immunohistochemistry	Used to detect LC3-II changes or LC3 puncta; a core antibody for autophagy detection
Ab112866	Recombinant LC3B Antibody	Recombinant, ExactAb™, Validated, See COA	LC3 detection	Used for LC3B protein expression, localization, and autophagosome formation analysis
Ab326883	Recombinant MAP1LC3B Antibody	KD Validation	LC3 detection	Suitable for validating antibody specificity in MAP1LC3B knockdown systems
M1465490	MAP1LC3B Human Pre-designed siRNA Set A		Autophagy-dependence validation	Used to knock down LC3B and validate LC3-related phenotypes
M1476126	MAP1LC3B2 Human Pre-designed siRNA Set A		Autophagy-dependence validation	Used for LC3B2-related autophagy mechanism studies
EJ1514111	Human Microtubule-associated Protein Light Chain 3B(LC3B) ELISA Kit	BioReagent	LC3B quantification	Used to detect LC3B levels in human-derived samples
EJ1512066	Rat Microtubule-Associated Proteins 1A/1B Light Chain 3B (MAP1LC3b) ELISA Kit	BioReagent	LC3B quantification	Used to detect LC3B levels in rat samples
A743649	Ad-GFP-p62		Autophagic substrate localization	Used to observe p62 aggregation, substrate recognition, and selective autophagy-related localization
A743657	Ad-mCherry-p62		Autophagic substrate localization	Used to observe p62-positive aggregates and autophagic substrate localization
P748883	pCMV-GFP-p62		Autophagic substrate localization	Used for GFP-p62 expression and p62 aggregate imaging
P748886	pCMV-mCherry-p62		Autophagic substrate localization	Used to observe p62 selective autophagy substrate localization
S1490309	SQSTM1 Human Pre-designed siRNA Set A		p62-dependence validation	Used to knock down p62 and validate selective autophagy-related mechanisms
Ab129090	SQSTM1 Mouse mAb	Carrier Free, ExactAb™, Validated, High Performance, See COA	Autophagic substrate degradation detection	Used to detect p62 levels by Western blot, immunofluorescence, or immunohistochemistry
EJ1514216	Human Sequestosome 1 (SQSTM1) ELISA Kit	BioReagent	p62 quantification	Used to detect p62/SQSTM1 levels in human-derived samples
EJ1512094	Rat Sequestosome 1 (SQSTM1) ELISA Kit	BioReagent	p62 quantification	Used to detect p62/SQSTM1 levels in rat samples

Table 11 Beclin 1-, ATG-, and ULK1-Related Detection Tools

Cat. No.	Product Name	Grade/Specification	Applicable detection stage	Application positioning
Ab215027	Beclin 1 Mouse mAb	Carrier Free,ExactAb™,Azide Free,Validated,High Performance,PBS Only,≥95%(SDS-PAGE),1.0 mg/mL	Autophagy initiation detection	Used to detect Beclin 1, a protein related to autophagy initiation and membrane nucleation
Ab326388	Recombinant Beclin 1 Antibody	KD Validation	Autophagy initiation detection	Suitable for detecting Beclin 1 signals in knockdown validation systems
Ab325630	Recombinant Beclin 1 Antibody	KD Validation	Autophagy initiation detection	Used for Beclin 1 expression and autophagy initiation analysis
Ab090924	Recombinant Beclin 1 Antibody	Recombinant, ExactAb™, Validated, See COA	Autophagy initiation detection	Used to detect Beclin 1 by Western blot or immunostaining
EJ1514449	Human Phosphorylated Serine/threonine-Protein kinase ULK1 (P-ULK1) ELISA Kit	BioReagent	Autophagy initiation signal detection	Used to quantify ULK1 phosphorylation and reflect autophagy initiation regulation
EJ1514781	Human Autophagy-related Protein 3 (ATG3) ELISA Kit	BioReagent	Autophagosome formation-related detection	Used to detect ATG3 levels and assist evaluation of LC3 lipidation-related processes
Ab326060	ATG5 Antibody	KD Validation	Autophagosome formation detection	Used to detect ATG5 and classical autophagosome formation mechanisms
Ab089177	Recombinant APG5L/ATG5 Antibody	ExactAb™, Validated, Recombinant, 0.3 mg/mL	Autophagosome formation detection	Used for ATG5 expression detection and autophagy-dependence validation
Ab326888	Recombinant ATG5 Antibody	KD Validation	Autophagosome formation detection	Suitable for ATG5 detection in knockdown validation contexts
A1476932	ATG5 Human Pre-designed siRNA Set A		Autophagy-dependence validation	Used to knock down ATG5 and validate dependence on the classical autophagy pathway
P743975	pLenti-ATG5-sgRNA		Antibody validation/mechanism control	Used for ATG5 antibody validation and negative controls
P743976	pLenti-ATG5-sgRNA		RNA detection control	Used as a transcriptional detection control in an ATG5 knockout background
EJ1514782	Human Autophagy Related Protein 5 (ATG5) ELISA Kit	BioReagent	Autophagosome formation-related detection	Used to detect ATG5 levels in human-derived samples
Ab090020	ATG7 Antibody	ExactAb™, Validated, 0.6 mg/mL	Autophagosome formation detection	Used to detect ATG7 expression and analyze LC3 lipidation-related mechanisms
Ab327383	Recombinant ATG7 Antibody	KD Validation	Autophagosome formation detection	Suitable for antibody validation in ATG7 knockdown systems
Ab327410	Recombinant ATG7 Antibody	KD Validation	Autophagosome formation detection	Used for ATG7-related autophagy mechanism studies
A1491055	ATG7 Human Pre-designed siRNA Set A		Autophagy-dependence validation	Used to knock down ATG7 and validate classical autophagy dependence

Table 12 Lysosomal Markers, Proteases, and Mitophagy-Related Tools

Cat. No.	Product Name	Grade/Specification	Applicable detection stage	Application positioning
Ab326444	LAMP1 /CD107a Antibody	KD Validation	Lysosomal localization/autophagy fusion	Used to detect the lysosomal membrane marker LAMP1
Ab187900	LAMP1 Mouse mAb	Carrier Free,ExactAb™,Azide Free,Validated,≥95%(SDS-PAGE),1.0 mg/mL	Lysosomal localization	Used for LC3-LAMP1 colocalization and lysosome number analysis
Ab112729	LAMP1/CD107a Mouse mAb	See COA	Lysosomal localization	Used for analysis of autophagosome-lysosome fusion
L1482144	LAMP1 Human Pre-designed siRNA Set A		Lysosomal function validation	Used to knock down LAMP1 and analyze lysosomal/autophagic fusion stages
P747584	pLenti-LAMP1-sgRNA		Antibody validation/mechanism control	Used for LAMP1 antibody validation and negative controls
P747585	pLenti-LAMP1-sgRNA		RNA detection control	Used for transcriptional detection in a LAMP1 knockout background
Ab112746	Recombinant LAMP2 Antibody	ExactAb™, Validated, Recombinant, 0.8 mg/mL	Lysosomal function detection	Used for LAMP2 expression, localization, and lysosome-related autophagic degradation analysis
Ab325845	Recombinant LAMP2 Antibody	KD Validation	Lysosomal function detection	Suitable for detection in LAMP2 knockdown validation contexts
L1484318	LAMP2 Human Pre-designed siRNA Set A		Lysosomal function validation	Used to validate LAMP2-dependent autophagosome-lysosome function
EJ1514271	Human Lysosomal Associated Membrane Protein 2 (LAMP2) ELISA Kit	BioReagent	Lysosomal marker quantification	Used to detect LAMP2 levels in human-derived samples
Ab326095	Cathepsin D LC Antibody	KD Validation	Lysosomal degradation function	Used to detect lysosomal protease-related degradation capacity
Ab215111	Cathepsin D Mouse mAb	Carrier Free,ExactAb™,Azide Free,Validated,High Performance,PBS Only,≥95%(SDS-PAGE),1.0 mg/mL	Lysosomal protease detection	Used for Cathepsin D expression and maturation analysis
C659428	Cathepsin D and E FRET Substrate acetate	≥98%	Lysosomal protease activity	Used for Cathepsin D/E enzyme activity detection and auxiliary evaluation of lysosomal degradation function
C1441538	Cathepsin D/E Substrate, Fluorogenic		Lysosomal protease activity	Used for fluorescence-based detection of Cathepsin D/E activity
P1491057	PINK1 Human Pre-designed siRNA Set A		Mitophagy validation	Used to knock down PINK1 and validate PINK1-dependent mitophagy
EJ1513577	Human PTEN Induced Putative Kinase 1 (PINK1) ELISA Kit	BioReagent	Mitophagy marker quantification	Used to detect PINK1 levels in human-derived samples
EJ1511892	Rat PTEN Induced Putative Kinase 1 (PINK1) ELISA Kit	BioReagent	Mitophagy marker quantification	Used to detect PINK1 levels in rat samples
EJ1512535	Mouse PTEN-induced Putative Kinase 1(PINK1) ELISA Kit	BioReagent	Mitophagy marker quantification	Used to detect PINK1 levels in mouse samples
Ab120363	Recombinant Parkin Antibody	Recombinant, ExactAb™, Validated, See COA	Mitophagy detection	Used for PINK1/Parkin pathway-related mitophagy studies
EJ1513570	Human Parkin Protein(PP) ELISA Kit	BioReagent	Mitophagy marker quantification	Used to detect Parkin protein levels in human-derived samples
C1509561	CCCP solution (10mmol/L)	BioReagent,10 mmol/L	Mitophagy induction	Used to induce decreased mitochondrial membrane potential and establish PINK1/Parkin-dependent mitophagy models
C424728	Carbonyl cyanide 3-chlorophenylhydrazone	10mM in DMSO	Mitophagy induction	Used for mitochondrial depolarization and mitophagy induction
C303974	Carbonyl cyanide 3-chlorophenylhydrazone (CCCP)	Moligand™, ≥98%	Mitophagy induction	Used for PINK1/Parkin pathway and mitochondrial clearance experiments
M1455700	MitoTracker Deep Red FM	≥99%	Mitochondrial localization/mitophagy	Used for mitochondrial mass, distribution, and colocalization analysis with LC3/LAMP1
M1455293	MitoTracker Orange CMTMRos		Mitochondrial localization/membrane potential-related observation	Used for mitochondrial structure and mitophagy-related colocalization analysis
N121291	Nile Red	BioReagent, for fluorescence analysis, ≥95%(HPLC)	Auxiliary lipophagy detection	Used for lipid droplet staining and can be combined with LC3/LAMP1 colocalization to analyze lipophagy
O421236	Oil red O	10mM in DMSO	Auxiliary lipophagy detection	Used for lipid accumulation observation; lipophagy interpretation requires autophagic flux indicators
O104972	Oil red O(C.1.26125)	Biological Stain	Auxiliary lipophagy detection	Used for lipid droplet and neutral lipid staining; suitable for lipophagy background observation

Cell autophagy detection should be designed around autophagic flux. LC3-II, p62, autophagy reporter systems, lysosomal function assays, and electron microscopy each reflect different stages. Only combined application can distinguish enhanced autophagy induction, autophagosome accumulation, and impaired lysosomal degradation.

For more related articles, please see below:

[1] Methods of observation and detection of cellular autophagy processes