Quantifying autophagosomes and autolysosomes in cells using imaging flow cytometry

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 LC31 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 of autophagy using amino acid starvation. This technique yields additional insight into autophagy processing making it a useful supplement to current techniques. V C 2015International Society for Advancement of Cytometry

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

“…Recent publications have emphasized the need to examine the concurrent formation of the autolysosome 2 17 20 21 . MIFC is uniquely able to measure the formation of the autolysosome by imaging the co-localization of the autophagosome to the lysosome 15 - 17 20 21 22 23 24 25 26 . In addition, the co-localization of p62 and LC3 using MIFC has also been explored to measure autophagic flux 5 .…”

“…The coefficient is log-transformed to increase the range to between zero and infinity 26 27 . BDS alone may not be sufficient; Rajan et al found that using only BDS could lead to false-positive or false-negative results 21 . BDS evaluates the co-localization of two markers of autophagy but does not consider the number of autophagosomes.…”

“…BDS evaluates the co-localization of two markers of autophagy but does not consider the number of autophagosomes. To account for the number of autophagosomes, Rajan et al included spot-counting of the LC3 puncta 21 . Rajan et al proposed using a bivariate scatter plot of LC3 spot count versus BDS.…”

Start of the new year's note, 2017—In the wake of the Rooster

“…He received this highest honor for his discoveries of mechanisms for autophagy (nobelprize.org; ). This is a good sign for Cytometry that did play a relevant role in his discoveries .…”

Apoptosis and autophagy

“…These regulatory processes are often studied in cancer cells because they require staged intracellular mechanisms, which if fully understood can be exploited as therapeutic targets. Cytometry as a multiparametric tool has been employed for decades to study these mechanisms (see a few recent publications on these subjects in References ), and the publications we highlight herein exemplify modern approaches toward that end.…”