title: Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy.
abstract: BACKGROUND
  Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved.
  
  
  METHODS
  Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties.
  
  
  RESULTS
  The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i) mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii) retention and spatial localization of chemical compounds vary within and between each cell line; and (iii) the structural similarities of compounds can infer their non-specific binding properties.
  
  
  CONCLUSIONS
  We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.
authors.affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Nath
authors.forename: Virginia A
authors.initials: VA
authors.lastname: Spencer
authors.forename: Ju
authors.initials: J
authors.lastname: Han
authors.forename: Hang
authors.initials: H
authors.lastname: Chang
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.forename: Gerald V
authors.initials: GV
authors.lastname: Fontenay
authors.forename: Charles
authors.initials: C
authors.lastname: Anderson
authors.forename: Joel M
authors.initials: JM
authors.lastname: Hyman
authors.forename: Marit
authors.initials: M
authors.lastname: Nilsen-Hamilton
authors.forename: Young-Tae
authors.initials: YT
authors.lastname: Chang
authors.forename: Bahram
authors.initials: B
authors.lastname: Parvin
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D008853:Microscopy / Q000379:methods*; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D054852:Small Molecule Libraries