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Related Experiment Video

Updated: Mar 6, 2026

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High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy.

J M Bravo-San Pedro1, F Pietrocola1, V Sica2

  • 1Gustave Roussy Cancer Campus, Villejuif, France; INSERM, U1138, Paris, France; Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie/Paris VI, Paris, France.

Methods in Enzymology
|March 4, 2017
PubMed
Summary

This study introduces a robust method for quantifying GFP-LC3 dots using automated fluorescence microscopy. This technique aids in screening for macroautophagy inducers and inhibitors by analyzing autophagosome formation.

Keywords:
CancerHigh-throughput screeningImageXpress Micro XLS Widefield High-Content Analysis SystemLysosomal inhibitorsNutrient deprivationRapamycin

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Macroautophagy is a crucial cellular process involving autophagosomes for degradation.
  • Microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), or LC3, accumulates in autophagosomes, serving as a marker.
  • Green fluorescent protein (GFP)-tagged LC3 (GFP-LC3) and fluorescence microscopy are used to monitor macroautophagy.

Purpose of the Study:

  • To develop a simple and robust method for high-throughput quantification of GFP-LC3 dots.
  • To facilitate the screening of chemical and biological libraries for autophagy modulators.
  • To accurately discriminate between increased autophagic flux and arrested autophagic degradation.

Main Methods:

  • Utilizing cell lines engineered to stably express GFP-LC3.
  • Employing automated fluorescence microscopy for high-throughput imaging.
  • Implementing automated image analysis for GFP-LC3 dot quantification.

Main Results:

  • A straightforward and robust method for quantifying GFP-LC3 puncta was established.
  • The method enables high-throughput analysis of macroautophagy.
  • This approach allows for the identification of autophagy inducers and inhibitors.

Conclusions:

  • Automated quantification of GFP-LC3 dots provides a powerful tool for autophagy research.
  • This method supports large-scale screening for compounds affecting macroautophagy.
  • Accurate measurement of autophagosome accumulation is key to understanding autophagy regulation.