Quantifying autophagosomes and autolysosomes in cells using imaging flow cytometry.
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 of autophagy using amino acid starvation. This technique yields additional insight into autophagy processing making it a useful supplement to current techniques.
School of Pharmacy, Department of Immunotherapeutics and Biotechnology, Texas Tech University Health Sciences Center, Abilene, Texas.
Robin
R
Rajan
Magdalena
M
Karbowniczek
Haley R
HR
Pugsley
Manoj K
MK
Sabnani
Aristotelis
A
Astrinidis
Ninh M
NM
La-Beck
D001343:Autophagy; D045744:Cell Line, Tumor; D058948:Cell Tracking; D002738:Chloroquine / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008247:Lysosomes / Q000473:pathology; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism; D010588:Phagosomes / Q000473:pathology
C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins; D002738:Chloroquine
LAMP1; LC3; autolysosome; autophagosome; autophagy; imaging flow cytometry; spot count