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Related Experiment Video

Updated: Jun 25, 2026

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells
09:34

The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Published on: July 27, 2018

Sequestration assays for mammalian autophagy.

Per O Seglen1, Anders Øverbye, Frank Saetre

  • 1Proteomics Section DNR, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway.

Methods in Enzymology
|February 10, 2009
PubMed
Summary

Measuring macroautophagic activity relies on cargo sequestration assays. These assays track long-lived proteins or probes, like lactate dehydrogenase (LDH), as they move into cellular organelles, indicating autophagic sequestration.

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Published on: July 21, 2017

Study of Protein-protein Interactions in Autophagy Research
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Study of Protein-protein Interactions in Autophagy Research

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Last Updated: Jun 25, 2026

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells
09:34

The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Published on: July 27, 2018

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry
11:39

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

Published on: July 21, 2017

Study of Protein-protein Interactions in Autophagy Research
14:08

Study of Protein-protein Interactions in Autophagy Research

Published on: September 9, 2017

Area of Science:

  • Cell Biology
  • Autophagy Research
  • Biochemical Assays

Background:

  • Macroautophagy is a fundamental cellular process for degrading long-lived proteins and organelles.
  • Accurate measurement of macroautophagic activity is crucial for understanding cellular health and disease.
  • Existing methods for assessing autophagic sequestration can be complex and require subcellular fractionation.

Purpose of the Study:

  • To describe and validate cargo sequestration assays for precise measurement of macroautophagic activity.
  • To introduce novel variants of cargo assays using protein fragments and exogenous probes for enhanced sensitivity and simplicity.
  • To enable background-free assessment of autophagic-lysosomal pathway function in whole cells.

Main Methods:

  • Utilized endogenous probes, such as lactate dehydrogenase (LDH), measuring its transfer from the cytosol to sedimentable organelle fractions.
  • Employed electrodisruption and Nycodenz density cushion centrifugation for efficient organelle separation.
  • Developed assays using intravacuolar protein fragments (e.g., p10(BHMT)) and exogenous probes (e.g., raffinose, lactose) for whole-cell analysis.

Main Results:

  • Demonstrated that cargo sequestration assays, particularly those using LDH, accurately reflect macroautophagic activity.
  • Showcased the utility of autophagic fragment assays (e.g., p10(BHMT) accumulation) for measuring autophagy in whole cells without fractionation.
  • Validated exogenous probes like raffinose for general autophagy measurement and lactose for dissecting autophagic-lysosomal pathway steps and interactions.

Conclusions:

  • Cargo sequestration assays provide a precise and direct method for quantifying macroautophagic activity.
  • Protein fragment and exogenous probe-based assays offer convenient, background-free alternatives for measuring autophagy in various cellular contexts.
  • These refined assays facilitate deeper understanding of the autophagic-lysosomal pathway's role in cellular homeostasis and disease.