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Related Concept Videos

Flow Cytometry01:23

Flow Cytometry

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The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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A Quantitative Flow Cytometry-Based Method for Autophagy Detection Across the Cell Cycle.

Vitaliy O Kaminskyy1

  • 1Department of Physiology and Pharmacology, Biomedicum, Karolinska Institutet, Stockholm, Sweden. Vitaly.Kaminsky@ki.se.

Methods in Molecular Biology (Clifton, N.J.)
|January 1, 2022
PubMed
Summary

This study presents a flow cytometry assay to measure autophagy, a key process in cancer, across the cell cycle. The method quantifies autophagosome-bound LC3 protein after selective membrane permeabilization.

Keywords:
Autophagic fluxAutophagosome formationAutophagyCell cycleCell permeabilizationDNA stainingFlow cytometryLC3Quantification

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Area of Science:

  • Cellular Biology
  • Cancer Research
  • Biotechnology

Background:

  • Autophagy is frequently deregulated in cancer.
  • Cancer cells often activate autophagy as a survival mechanism against therapies.
  • Quantifying autophagy across the cell cycle is crucial for understanding its role in cancer.

Purpose of the Study:

  • To develop and describe a flow cytometry-based assay for characterizing autophagy.
  • To enable the quantitative assessment of autophagy in relation to the cell cycle.
  • To provide a method for analyzing autophagy in live or fixed cells.

Main Methods:

  • Selective plasma membrane permeabilization using digitonin.
  • Extraction of membrane-unbound LC3 protein.
  • Staining of autophagosome-bound LC3 protein with a specific antibody.
  • DNA labeling with propidium iodide for cell cycle analysis.
  • Flow cytometry for quantitative detection.

Main Results:

  • The described method allows for the characterization of autophagy across the cell cycle.
  • The assay enables the quantification of autophagosome-bound LC3 protein.
  • The method can be combined with staining for other cellular markers.

Conclusions:

  • This flow cytometry assay provides a robust method for quantitative autophagy analysis.
  • The assay facilitates the study of autophagy dynamics throughout the cell cycle.
  • This technique can be integrated with other cellular process analyses for comprehensive insights into cancer biology.