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Protocol for Probing Regulated Lysosomal Activity and Function in Living Cells.

STAR protocols (2020-12-31)
L V Albrecht, N Tejeda-Muñoz, E M De Robertis
ABSTRACT

Lysosomes are the catabolic center of the cell. Limitations of many lysosomal tracers include low specificity and lack of reliable physiological readouts for changes in growth factor-regulated lysosomal activity. The imaging-based protocols described here provide insights at the cellular level to quantify functions essential to lysosomal biology, including β-glucosidase enzymatic cleavage, active Cathepsin D, and pH regulation in real time. These optimized protocols, applied in different cell types and pathophysiologic contexts, provide useful tools to study lysosome function in cultured living cells. For complete details on the use and execution of this protocol, please refer to Albrecht et al. (2020).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride, ACS reagent, ≥99.0%
Sigma-Aldrich
CHIR99021, ≥98% (HPLC)
Sigma-Aldrich
Collagen from rat tail, Bornstein and Traub Type I, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
5-(N-Ethyl-N-isopropyl)amiloride
Sigma-Aldrich
IPA-3, ≥95% (HPLC), powder