Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow...

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Autores principales:	Michael Degtyarev, Mike Reichelt, Kui Lin
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4a2b0d377fd54403b86f84c3184b5089
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spelling	oai:doaj.org-article:4a2b0d377fd54403b86f84c3184b50892021-11-18T08:34:56ZNovel quantitative autophagy analysis by organelle flow cytometry after cell sonication.1932-620310.1371/journal.pone.0087707https://doaj.org/article/4a2b0d377fd54403b86f84c3184b50892014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24489953/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. 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. 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. 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. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.Michael DegtyarevMike ReicheltKui LinPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 1, p e87707 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Michael Degtyarev Mike Reichelt Kui Lin Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.
description	Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. 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. 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. 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. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.
format	article
author	Michael Degtyarev Mike Reichelt Kui Lin
author_facet	Michael Degtyarev Mike Reichelt Kui Lin
author_sort	Michael Degtyarev
title	Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.
title_short	Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.
title_full	Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.
title_fullStr	Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.
title_full_unstemmed	Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.
title_sort	novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/4a2b0d377fd54403b86f84c3184b5089
work_keys_str_mv	AT michaeldegtyarev novelquantitativeautophagyanalysisbyorganelleflowcytometryaftercellsonication AT mikereichelt novelquantitativeautophagyanalysisbyorganelleflowcytometryaftercellsonication AT kuilin novelquantitativeautophagyanalysisbyorganelleflowcytometryaftercellsonication
_version_	1718421619302465536

Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.

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