Abstract
Autophagy dysregulation has been implicated in numerous diseases and many therapeutic agents are known to modulate this pathway. Therefore, the ability to accurately monitor autophagy is critical to understanding its role in the pathogenesis and treatment of many diseases. Recently an imaging flow cytometry method measuring colocalization of microtubule associated protein 1B light chain 3 (LC3) and lysosomal signals via Bright Detail Similarity (BDS) was proposed which enabled evaluation of autophagic processing. However, since BDS only evaluates colocalization of LC3 and lysosomal signals, the number of autophagy organelles was not taken into account. We found that in cells classified as having Low BDS, there was a large degree of variability in accumulation of autophagosomes. Therefore, we developed a new approach wherein BDS was combined with number of LC3+ puncta, which enabled us to distinguish between cells having very few autophagy organelles versus cells with accumulation of autophagosomes or autolysosomes. Using this method, we were able to distinguish and quantify autophagosomes and autolysosomes in breast cancer cells cultured under basal conditions, with inhibition of autophagy using chloroquine, and with induction ...Continue Reading
References
Apr 8, 2004·Developmental Cell·Beth Levine, Daniel J Klionsky
May 26, 2004·Brain Research·Kazuhiro NakasoKenji Nakashima
Nov 6, 2004·Science·Takahiro Shintani, Daniel J Klionsky
Jul 6, 2007·Autophagy·Noboru Mizushima, Tamotsu Yoshimori
May 5, 2009·Autophagy·David C RubinszteinDaniel J Klionsky
Aug 6, 2009·Annual Review of Genetics·Congcong He, Daniel J Klionsky
Oct 20, 2009·European Journal of Pharmacology·V Raja Solomon, Hoyun Lee
Feb 4, 2010·The Journal of Biological Chemistry·You-Tong WuHan-Ming Shen
Feb 11, 2010·Cell·Noboru MizushimaBeth Levine
May 4, 2011·The Journal of Biological Chemistry·Natura Myeku, Maria E Figueiredo-Pereira
May 25, 2011·Autophagy·Claire de la CalleMatthew L Albert
Feb 4, 2012·Autophagy·Kanchan PhadwalAnna Katharina Simon
Sep 12, 2012·Autophagy·Daniel J KlionskyBrian Zuckerbraun
Mar 26, 2013·Acta Pharmacologica Sinica·Ya-ping YangChun-feng Liu
Citations
Nov 10, 2015·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Tobias BraunAgnes Csiszár
Jun 6, 2016·Methods : a Companion to Methods in Enzymology·Haley R Pugsley
Sep 14, 2016·Methods : a Companion to Methods in Enzymology·Brian K McFarlin, Melody A Gary
Jan 21, 2017·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Attila Tárnok
Jun 29, 2017·Cells·Fengjuan WangSylviane Muller
Oct 24, 2017·Toxins·Sarah J C WhitfieldGraeme C Clark
Jun 18, 2019·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Jessica P Houston
Jan 18, 2019·Cancers·Julianne D Twomey, Baolin Zhang
May 7, 2020·Cells·Dar-Shong LinMing-Fu Chiang
Mar 7, 2021·Cell Death & Disease·Carolyn N BrownCharles L Edelstein
Jun 25, 2021·PloS One·Joseph A SebastianMichael C Kolios
Nov 14, 2017·Chemical Research in Toxicology·Mariangela LibrizziClaudio Luparello