A versatile, bar-coded nuclear marker/reporter for live cell fluorescent and multiplexed high content imaging

PLoS One. 2013 May 14;8(5):e63286. doi: 10.1371/journal.pone.0063286. Print 2013.


The screening of large numbers of compounds or siRNAs is a mainstay of both academic and pharmaceutical research. Most screens test those interventions against a single biochemical or cellular output whereas recording multiple complementary outputs may be more biologically relevant. High throughput, multi-channel fluorescence microscopy permits multiple outputs to be quantified in specific cellular subcompartments. However, the number of distinct fluorescent outputs available remains limited. Here, we describe a cellular bar-code technology in which multiple cell-based assays are combined in one well after which each assay is distinguished by fluorescence microscopy. The technology uses the unique fluorescent properties of assay-specific markers comprised of distinct combinations of different 'red' fluorescent proteins sandwiched around a nuclear localization signal. The bar-code markers are excited by a common wavelength of light but distinguished ratiometrically by their differing relative fluorescence in two emission channels. Targeting the bar-code to cell nuclei enables individual cells expressing distinguishable markers to be readily separated by standard image analysis programs. We validated the method by showing that the unique responses of different cell-based assays to specific drugs are retained when three assays are co-plated and separated by the bar-code. Based upon those studies, we discuss a roadmap in which even more assays may be combined in a well. The ability to analyze multiple assays simultaneously will enable screens that better identify, characterize and distinguish hits according to multiple biologically or clinically relevant criteria. These capabilities also enable the re-creation of complex mixtures of cell types that is emerging as a central area of interest in many fields.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biomarkers / metabolism
  • Cell Nucleus / metabolism*
  • Cell Proliferation
  • Cell Survival
  • Coculture Techniques
  • HeLa Cells
  • Humans
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence / methods*
  • Molecular Imaging / methods*
  • Molecular Sequence Data
  • Nuclear Localization Signals


  • Biomarkers
  • Luminescent Proteins
  • Nuclear Localization Signals