RRC ID 47684
Author Frost LS, Dhingra A, Reyes-Reveles J, Boesze-Battaglia K.
Title The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo.
Journal Methods Enzymol
Abstract There is increasing evidence documenting the critical role played by autophagic and autophagy-associated processes in maintaining cell homeostasis and overall systemic health. Autophagy is considered a degradative as well as a recycling pathway that relies on encapsulated intracellular components trafficking to and fusing with degradative compartments, including lysosomes. In this chapter, we describe the use of DQ™-BSA to study autophagosome-lysosome fusion as well as a means by which to analyze hybrid autophagic pathways. Such noncanonical pathways include LC3-associated phagocytosis, better known as LAP. Both autophagosomes and LAPosomes (LC3-associated phagosomes) deliver cargo for degradation. The use of fluorescent DQ™-BSA in conjugation with autophagic makers and biomarkers of hybrid autophagy offers a reliable technique to monitor the formation of autolysosomes and LAPo-lysosomes in both fixed- and live-cell studies. This technique relies on cleavage of the self-quenched DQ™ Green- or DQ™ Red BSA protease substrates in an acidic compartment to generate a highly fluorescent product.
Volume 587
Pages 43-54
Published 2016-11-17
DOI 10.1016/bs.mie.2016.09.052
PII S0076-6879(16)30325-1
PMID 28253971
PMC PMC5338641
MeSH Autophagosomes / metabolism Autophagy* Boron Compounds / chemistry Cell Line Epithelial Cells / metabolism Fluorescent Dyes / chemistry Fluorescent Dyes / metabolism Humans Lysosomes / metabolism Microscopy, Confocal / methods Microtubule-Associated Proteins / analysis Microtubule-Associated Proteins / metabolism* Molecular Biology / methods* Serum Albumin, Bovine / chemistry Serum Albumin, Bovine / metabolism*
IF 1.394
Times Cited 17
Mice RBRC00806