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Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy
Nath, S Spencer, VA Han, J Chang, H Zhang, K Fontenay, GV Anderson, C Hyman, JM Nilsen-Hamilton, M Chang, YT
Nath, S
Spencer, VA
Han, J
Chang, H
Zhang, K
Fontenay, GV
Anderson, C
Hyman, JM
Nilsen-Hamilton, M
Chang, YT
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Journal Article
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2012-01-05
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10.1371/journal.pone.0028802
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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. © 2012 Nath et al.
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