Measurement of Autophagy by Flow Cytometry

Gary Warnes1

1 Flow Cytometry Core Facility, Blizard Institute, Barts & The London School of Medicine and Dentistry, Queen Mary London University, London
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.45
DOI:  10.1002/0471142956.cy0945s68
Online Posting Date:  April, 2014
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In recent years, flow cytometry has been used to detect the presence of autophagy mainly by the fluorescent antibody labeling of the autophagy marker, the microtubule associated protein LC3‐II. Here we describe the indirect antibody labeling of LC3‐II in cells displaying drug‐induced autophagy by the use of rapamycin and chloroquine, as well as cells undergoing serum starvation. Although the mechanism of action of LysoTracker dyes is not fully understood, lysosomal mass increases during the autophagic process to enable the cell to produce autolysosomes. Given that LC3‐II and LysoTracker are measuring different biological events in the autophagic process, they surprisingly both up‐regulated during autophagic process. This approach shows that although LysoTracker dyes do not specifically label lysosomes or autophagosomes within the cell, they allow the simultaneous measurement of an autophagy related process and other live cell functions, which is not possible with the standard LC3‐II antibody technique. Curr. Protoc. Cytom. 68:9.45.1‐9.45.10. © 2014 by John Wiley & Sons, Inc.

Keywords: autophagy; LC3‐II; Lysotracker; lysosomes; autophagosomes; autolysosomes

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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Autophagy by Intracellular Labeling with Anti‐LC3‐II
  • Alternate Protocol 1: LysoTracker Labeling of Autophagic Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Detection of Autophagy by Intracellular Labeling with Anti‐LC3‐II

  • Jurkat T cells (European Collection of Cell Cultures)
  • K562 erythromyeloid cells (European Collection of Cell Cultures)
  • RPMI 1640 with glutamine (Life Technologies)
  • Fetal bovine serum (FBS; Life Technologies)
  • 200 U/ml penicillin/200 µg/ml streptomycin solution
  • Chloroquine (CQ; see recipe)
  • Rapamycin (see recipe)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 14190‐094)
  • Caltag Fix Solution A
  • 5% (v/v) Triton X‐100 solution (Sigma Aldrich; final concentration 0.25%)
  • Polyclonal LC3II rabbit antibody, 1 µg/µl (see recipe))
  • 2 µg/µl rabbit immunoglobulin, (see recipe)
  • 2 mg/µl goat‐anti‐rabbit Alexa Fluor‐647 (see recipe)
  • 75‐cm2 tissue culture flask
  • Tissue culture (TC) hood
  • 50‐ and 15‐ml conical tubes
  • Thermo Multifuge 3SR+ centrifuge
  • CO 2 incubator at 37°C
  • 12 × 75–mm polystyrene tubes (Falcon)
  • Vortex mixer
  • Flow cytometer with red laser light source

Alternate Protocol 1: LysoTracker Labeling of Autophagic Cells

  Additional Materials (see Basic Protocol)
  • LysoTracker Green (LTG; see recipe)
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