Development of a Kinetic Assay for Late Endosome Movement

J Biomol Screen. 2014 Aug;19(7):1070-8. doi: 10.1177/1087057114524278. Epub 2014 Feb 20.


Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

Keywords: Lamp1; cardiac glycoside; high-content imaging; live cell; tracking.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Automation
  • Cell Line, Tumor
  • Cell Migration Assays / methods*
  • Combinatorial Chemistry Techniques
  • Endosomes / metabolism*
  • Green Fluorescent Proteins / chemistry
  • Humans
  • Image Processing, Computer-Assisted
  • Microscopy, Fluorescence
  • Movement
  • Multivariate Analysis
  • Nocodazole / chemistry
  • Phenotype
  • Principal Component Analysis
  • RNA Interference
  • Reproducibility of Results


  • Green Fluorescent Proteins
  • Nocodazole