A live-cell fluorescence microplate assay suitable for monitoring vacuolation arising from drug or toxic agent treatment

J Biomol Screen. 2010 Apr;15(4):398-405. doi: 10.1177/1087057110364242. Epub 2010 Mar 17.


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.

MeSH terms

  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Chloroquine / pharmacology
  • Drug Evaluation, Preclinical / instrumentation*
  • Drug Evaluation, Preclinical / methods*
  • Humans
  • Luminescent Measurements / instrumentation
  • Luminescent Measurements / methods*
  • Macrolides / pharmacology
  • Microscopy, Fluorescence
  • Pharmaceutical Preparations / analysis*
  • Phospholipids / metabolism
  • Small Molecule Libraries / analysis
  • Small Molecule Libraries / toxicity*
  • Vacuoles / drug effects*
  • Vacuoles / metabolism*


  • Macrolides
  • Pharmaceutical Preparations
  • Phospholipids
  • Small Molecule Libraries
  • Chloroquine
  • bafilomycin A1