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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Related Experiment Video

Updated: Jul 1, 2025

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond
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Real-Time Autophagic Flux Measurements in Live Cells Using a Novel Fluorescent Marker DAPRed.

Arnold Sipos1,2, Kwang-Jin Kim1,2,3,4,5, Juan R Alvarez1,2

  • 1Will Rogers Institute Pulmonary Research Center and Hastings Center for Pulmonary Research, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

Bio-Protocol
|March 11, 2024
PubMed
Summary

This study introduces DAPRed, a novel live-cell imaging method to measure autophagic flux in real time. This technique offers a straightforward way to visualize and quantify autophagy kinetics in single cells, aiding disease research.

Keywords:
A549 cellsAutophagic fluxConfocal microscopyDAPRedLive cell imaging

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Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy
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Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Autophagy is a fundamental cellular process crucial for maintaining homeostasis and implicated in various diseases.
  • Existing methods for studying autophagy, such as western blotting, lack the temporal resolution needed to assess dynamic autophagic flux.
  • Understanding autophagy kinetics is vital for insights into diseases like Alzheimer's and cardiac conditions.

Purpose of the Study:

  • To develop and validate a novel method for real-time measurement of autophagic flux.
  • To enable visualization and quantification of autophagy dynamics at the single-cell level.
  • To provide a more accessible technique for studying autophagy in various cell models.

Main Methods:

  • Development of DAPRed, a novel fluorescent probe for autophagic flux measurement.
  • Utilizing live-cell imaging with confocal microscopy on A549 cells.
  • Real-time visualization and quantification of autophagic flux in single live cells.

Main Results:

  • DAPRed allows for real-time, qualitative imaging of autophagic flux at the single-cell level.
  • The technique is applicable to both cell lines and primary cells.
  • Confocal microscopy enables visualization of autophagy without disrupting cellular functions.

Conclusions:

  • DAPRed provides a straightforward and effective approach for assessing time-dependent autophagy kinetics.
  • This novel method facilitates deeper understanding of autophagy's role in cellular processes and disease.
  • The technique's adaptability makes it valuable for diverse in vitro cell and tissue models.