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Related Experiment Video

Updated: Mar 7, 2026

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Published on: July 21, 2017

Renilla Luciferase-LC3 Based Reporter Assay for Measuring Autophagic Flux.

T Farkas¹, M Jäättelä¹

¹Cell Death and Metabolism, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, Copenhagen, Denmark.

Methods in Enzymology

|February 27, 2017

Summary

We developed a new assay to measure macroautophagy (autophagy) flux by tracking Rluc-LC3 protein degradation. This method efficiently screens compounds and genes affecting autophagy in cells.

Keywords:

Autophagy High-throughput screening assay LC3 Luciferase Real time Signaling

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Last Updated: Mar 7, 2026

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

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Published on: July 21, 2017

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Published on: April 28, 2023

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

Published on: September 7, 2008

Area of Science:

Cell Biology
Molecular Biology
Biochemistry

Background:

Macroautophagy (autophagy) is a fundamental cellular process for degrading damaged components.
Accurately measuring autophagy flux is crucial for understanding its role in health and disease.
Existing methods for monitoring autophagy flux can be complex and time-consuming.

Purpose of the Study:

To develop a novel, indirect assay for quantifying autophagy flux.
To establish a luminescence-based method for high-throughput screening of autophagy regulators.
To provide a versatile tool for studying autophagy in various experimental settings.

Main Methods:

An indirect autophagy flux assay was established using a fusion protein, Rluc-LC3, which is associated with autophagosomes.
The assay monitors the degradation of Rluc-LC3 via luminescence detection.
The method is applicable to both cell lysates and live-cell imaging.

Main Results:

The Rluc-LC3 assay provides a reliable measure of autophagy flux.
The assay is suitable for screening large numbers of samples in parallel.
Demonstrated utility in identifying regulators of autophagy flux, including genes, miRNAs, and small molecules.

Conclusions:

The Rluc-LC3 luminescence assay offers an efficient and adaptable approach to measure macroautophagy flux.
This assay facilitates the discovery of novel modulators of the autophagy pathway.
The method is valuable for advancing research in autophagy-related cellular processes.