TY - JOUR T1 - Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy JF - PLoS One Y1 - 2012 A1 - Nath, Sangeeta A1 - Spencer, Virginia A A1 - Ju Han A1 - Hang Chang A1 - Zhang, Kai A1 - Fontenay, Gerald V A1 - Anderson, Charles A1 - Hyman, Joel M A1 - Nilsen-Hamilton, Marit A1 - Chang, Young-Tae A1 - Parvin, Bahram KW - Animals KW - Arabidopsis KW - Cell Line KW - Cell Survival KW - Combinatorial Chemistry Techniques KW - Fluorescent Dyes KW - Humans KW - Ligands KW - Mice KW - Microscopy KW - Small Molecule Libraries AB -

INTRODUCTION: 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.

METHOD: 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.

CONCLUSION: 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.

VL - 7 IS - 1 ER -