Lc3 Reporter
Lc3 Reporter - Related Videos
11:39
Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry
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Cited by 94 •
2017
Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.
06:58
Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles
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Cited by 7 •
2024
Here, we present the methodology for concisely assessing autophagosome marker LC3-II levels in extracellular vesicles (EVs) by immunoblotting. Analysis for LC3-II levels in EVs, autolysosome formation, and omegasome formation suggests the new role of STX6 in the release of LC3-II-positive EVs when autophagosome-lysosome fusion is inhibited.
01:24
Data Reporting and Recording
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2023
Reporting and recording are crucial in data documentation. The timely, thorough, and accurate documentation of facts is essential when recording patient data. Failure to record findings during an assessment or interpretation of a problem will result in loss of information and make the patient document unreliable. The reader is left with general impressions if the information is not specific. A recording is documenting data of the individual's health information in a traceable, secure, and...
08:07
Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence
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Cited by 3 •
2023
The goal of this protocol is to determine autophagic levels in pancreatic cancer and pancreatic acinar cells through LC3 immunofluorescence and LC3 dot quantification.
11:53
Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy
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Cited by 6 •
2023
Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.
FAQs
The reporter’s two signals separate an earlier membrane-associated state from a later lysosomal state. LC3-bearing autophagosomes retain both GFP and RFP before lysosomal fusion. Once fusion creates an acidic autolysosome, GFP is quenched while RFP remains detectable. Green-plus-red and red-only patterns therefore correspond to different stages of the pathway.
Flux interpretation depends on following the transition between these fluorescence states, not merely tallying LC3-positive structures. A high number of labeled vesicles could reflect increased autophagosome formation or incomplete delivery to lysosomes. The GFP-to-RFP change supplies evidence that structures have progressed to lysosomal fusion, making pathway activity more informative than a static vesicle count.
Acid sensitivity gives the reporter its stage-resolving capability. GFP signal decreases when the LC3 reporter reaches the acidic lysosomal environment, whereas RFP continues to provide a visible signal. This contrast allows investigators to recognize autolysosome formation even when the original green fluorescence is no longer present, linking fluorescence behavior to compartment conditions.
Interpretation should focus on color combinations rather than fluorescence intensity alone. Colocalized green and red signal is consistent with LC3 on autophagosomal membranes, while predominantly red signal indicates that those structures have undergone fusion with acidic lysosomes. This distinction helps separate pathway progression from simple accumulation of fluorescent puncta.
In biology, the reporter can connect autophagy dynamics with cellular stress and organelle quality control. It also provides a way to examine how disease-related cellular changes affect degradation pathways and how candidate therapeutics alter autophagic behavior. These applications make the tool useful for linking fluorescence patterns to broader cellular phenotypes.
At a conceptual level, use begins by examining the reporter’s green and red fluorescence, then assigning patterns to autophagosomes or autolysosomes according to LC3 membrane association, lysosomal fusion, and GFP quenching. Comparing these states provides an estimate of pathway progression and supports evaluation of stress, disease, or treatment-related changes.