Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy
Title | Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy |
Publication Type | Journal Article |
Year of Publication | 2012 |
Authors | Nath S, Spencer VA, Han J, Chang H, Zhang K, Fontenay GV, Anderson C, Hyman JM, Nilsen-Hamilton M, Chang Y-T, Parvin B |
Journal | PLoS One |
Volume | 7 |
Pagination | e28802 |
Date Published | 2012 |
ISSN | 1932-6203 |
Keywords | Animals, Arabidopsis, Cell Line, Cell Survival, Combinatorial Chemistry Techniques, Fluorescent Dyes, Humans, Ligands, Mice, Microscopy, Small Molecule Libraries |
Abstract | 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. |
DOI | 10.1371/journal.pone.0028802 |