A Quantitative Flow Cytometry-Based Method for Autophagy Detection Across the Cell Cycle

Vitaliy O Kaminskyy

doi:10.1007/978-1-0716-2071-7_5

A Quantitative Flow Cytometry-Based Method for Autophagy Detection Across the Cell Cycle

Methods Mol Biol. 2022:2445:65-74. doi: 10.1007/978-1-0716-2071-7_5.

Author

Vitaliy O Kaminskyy¹

Affiliation

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

PMID: 34972986
DOI: 10.1007/978-1-0716-2071-7_5

Abstract

Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry-based assay to characterize autophagy across the cell cycle is described. This method is based on selective plasma membrane permeabilization with digitonin and extraction of membrane-unbound LC3 protein followed by staining of the autophagosome-bound LC3 protein with antibody and labeling of DNA with propidium iodide. Staining with the LC3 antibody described here can be also combined with the staining of other cellular markers, allowing to quantitatively assess autophagy in relation to different cellular processes by flow cytometry.

Keywords: Autophagic flux; Autophagosome formation; Autophagy; Cell cycle; Cell permeabilization; DNA staining; Flow cytometry; LC3; Quantification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Autophagosomes / metabolism
Autophagy* / physiology
Cell Cycle
Flow Cytometry / methods
Microtubule-Associated Proteins* / metabolism

Substances

Microtubule-Associated Proteins