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Flow Cytometric Analysis of Autophagic Activity with Cyto-ID Staining in Primary Cells   

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Original research article

A brief version of this protocol appeared in:
Antimicrobial Agents and Chemotherapy
Jan 2013

Abstract

Flow cytometry allows very sensitive and reliable high-throughput analysis of autophagic flux. This methodology permits to screen cells in flow and capture multi-component images. Using this technology autophagic flux may be analysed accurately in both suspension as well as adherent cells upon trypsinization independent of how heterogeneous the autophagosomal content might be. The method is based on Cyto-ID staining of autophagic compartments (pre-autophagosomes, autophagosomes, and autophagolysosomes) in live cells using Cyto-ID® Autophagy Detection Kit. Autophagic compartments are intermediate constituents of a dynamic lysosomal degradation process and their intracellular abundance at a particular time point is a function of the established equilibrium between their generation and degradation. Determination of autophagic flux facilitates the discrimination between early induction of autophagosome formation and late inhibition of autophagosome maturation as both results in an ultimate increase in autophagosomal presence. Cyto-ID assay is based on the usage of a specific dye that selectively stains autophagic compartments and therefore allows determination of autophagic flux as accumulation of stained compartments in basic or activated conditions [rapamycin (1-5 µmol/L), PP242 (1-5 µmol/L) or Hanks’ Balanced Salt Solution containing 6 mmol/L glucose (starvation medium)] after blockage of autophagolysosomal degradation using lysosomotropic compounds such as ammonium chloride (NH4Cl) (10-20 mmol/L) or chloroquine (CQ) (5-10 µmol/L). ΔMFI Cyto-ID = MFI Cyto-ID (+CQ/NH4Cl) - MFI Cyto-ID (-CQ/NH4Cl).

Keywords: Autophagy, Primary Cells, Cyto-ID, Autophagic flux, Flow Cytometry

Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Stankov, M., Panayotova-Dimitrova, D., Leverkus, M., Klusmann, J. and Behrens, G. (2014). Flow Cytometric Analysis of Autophagic Activity with Cyto-ID Staining in Primary Cells. Bio-protocol 4(7): e1090. DOI: 10.21769/BioProtoc.1090.
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