Autophagy is a complex and dynamic process, which is challenging to measure accurately. Macroautophagy (autophagy hereafter) is a well-conserved cellular catabolic process of self-degradation through the lysosomal machinery, and plays an important role in both normal physiology and diseases. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. There are no patents, products in development or marketed products to declare. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Д¬ompeting interests: All authors are employees of Genentech, Inc., a member of the Roche group. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ДЇunding: This study was funded by Genentech, Inc., a member of the Roche group.

Received: AugAccepted: JanuPublished: January 29, 2014Д¬opyright: В© 2014 Degtyarev et al. PLoS ONE 9(1):Д®ditor: Alan Graham Pockley, Nottingham Trent University, United Kingdom This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.Д¬itation: Degtyarev M, Reichelt M, Lin K (2014) Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation.

These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes.

Flowjo 10 backgating fully gated