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Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

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Published on: April 28, 2023

Modified LC3 Dot Quantification Method.

Tahira Anwar¹, Eeva-Liisa Eskelinen²

¹Molecular and Integrative Biosciences Research Program, University of Helsinki, Helsinki, Finland.

Methods in Molecular Biology (Clifton, N.J.)

|January 1, 2022

Summary

Researchers developed a new method to quantify autophagy by measuring the proportion of vesicular LC3 protein staining. This technique enhances the precise monitoring of autophagic processes in cells.

Keywords:

Autophagy Cell profiler Fluorescence microscopy Image analysis Immunofluorescence LC3

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Published on: April 18, 2018

Area of Science:

Cell Biology
Molecular Biology
Biochemistry

Background:

Autophagy, a cellular degradation process, has revealed diverse physiological and pathological roles since the discovery of its genes and proteins in the early 1990s.
Accurate monitoring of autophagy is crucial for understanding its functions.
Western blotting and fluorescence microscopy targeting the LC3 marker protein are commonly employed methods for autophagy assessment.

Purpose of the Study:

To introduce a refined protocol for quantifying autophagy.
To improve the precision of monitoring autophagy by modifying the existing LC3 dot counting method.
To provide a method for assessing the proportion of vesicular LC3 staining relative to total LC3 staining within individual cells.

Main Methods:

Modification of the standard LC3 dot counting assay.
Quantification of the proportion of vesicular LC3 puncta within cells.
Utilizing fluorescence microscopy to analyze endogenous LC3 protein localization.

Main Results:

The developed protocol allows for the measurement of the ratio of vesicular LC3 puncta to total LC3 puncta per cell.
This method offers a more precise quantification of autophagy.
The approach is well-suited for analyzing endogenous LC3 protein levels.

Conclusions:

The modified LC3 dot counting method provides a robust approach for precise autophagy quantification.
This protocol enhances the ability to monitor autophagic flux by assessing LC3 vesicle formation.
The technique is valuable for research involving endogenous LC3 protein in various biological contexts.