A live-cell fluorescence microplate assay suitable for monitoring vacuolation arising from drug or toxic agent treatment.
Lysosomes are membrane-bound subcellular organelles involved in the degradation of macromolecules and pathogens in diverse processes, including endocytosis, phagocytosis, and autophagy. A red fluorescent probe was developed that is selectively sequestered in acidic organelles. U20S cells pretreated with 64 microM chloroquine for as little as 5 h show a dramatic increase in lysosome-like vesicle number and volume. The probe can be employed for highlighting lysosome-like organelles under conditions wherein cells produce vacuoles that contain most of the degradative enzymes of the lysosome but are not as acidic as the parent organelle. Using a conventional fluorescence microplate reader, the half-maximal effective concentration (EC(50)) of chloroquine was estimated. The high Z' score obtained using the assay demonstrated excellent signal-to-noise ratios. The fluorescence microplate assay was successfully employed to screen a small-molecule compound library for agents that increase lysosomal volume and number. One potential application of the new assay is in the toxicology portion of preclinical drug safety assessment (ADME-Tox) workflows, using in vitro cell culture models to aid in the drug development process.
Coleman
Jack
J
R&D, Enzo Life Sciences, Farmingdale, New York 11735, USA.
Xiang
Yuejun
Y
Pande
Praveen
P
Shen
Dee
D
Gatica
Divina
D
Patton
Wayne F
WF
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002738:Chloroquine / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D018942:Macrolides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D004364:Pharmaceutical Preparations / Q000032:analysis*; D010743:Phospholipids / Q000378:metabolism; D054852:Small Molecule Libraries / Q000032:analysis / Q000633:toxicity*; D014617:Vacuoles / Q000187:drug effects* / Q000378:metabolism*
D018942:Macrolides; D004364:Pharmaceutical Preparations; D010743:Phospholipids; D054852:Small Molecule Libraries; D002738:Chloroquine; C040929:bafilomycin A1