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Quantitating the concentration of Py-Im polyamide–fluorescein conjugates in live cells

Hsu, Carey F. and Dervan, Peter B. (2008) Quantitating the concentration of Py-Im polyamide–fluorescein conjugates in live cells. Bioorganic and Medicinal Chemistry Letters, 18 (22). pp. 5851-5855. ISSN 0960-894X. PMCID PMC2587135. https://resolver.caltech.edu/CaltechAUTHORS:HSUbmcl08

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Abstract

Quantitative fluorescence-based methods have been developed to determine the nuclear concentration of polyamide–fluorescein conjugates in cell culture. Confocal laser scanning microscopy and flow cytometry techniques are utilized to plot calibration curves, from which the nuclear concentration can be interpolated. Upon treatment with polyamide, the concentration in the nucleus of live HeLa cells is calculated to be between 0.1 and 0.5 μM, which is significantly lower than the 2 μM dosage concentration. In contrast, the observed nuclear concentration in U251 cells is closer to the dosage concentration, indicating a cell line-specific increase in uptake for this class of compounds. Although confocal microscopy and flow cytometry generate disparate values, taken together these experiments suggest that the polyamide concentration inside the cell nucleus is lower than it is outside the cell.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.bmcl.2008.05.063DOIArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2587135/UNSPECIFIEDArticle
ORCID:
AuthorORCID
Dervan, Peter B.0000-0001-8852-7306
Additional Information:© 2008 Elsevier. Received 10 April 2008; revised 14 May 2008; accepted 15 May 2008. Available online 20 May 2008. We thank Shelley Diamond and the Caltech Flow Cytometry Facility for instrumentation assistance. We also thank Scott Fraser and the Beckman Institute Biological Imaging Center for helpful discussions. We are grateful to the National Institutes of Health for research support. Mass spectrometry analyses were performed in the Mass Spectrometry Facility of the Division of Chemistry and Chemical Engineering at the California Institute of Technology. Fluorimetry measurements were performed in the Beckman Institute Laser Resource Center. Supplementary data: Quantitative DNase I footprint titration experiments and quantitative RT-PCR experiments (Supplementary Fig. S1), confocal laser scanning microscopy images (Supplementary Fig. S2), and experimental details (Supplementary Information). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2008.05.063.
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Funding AgencyGrant Number
NIHUNSPECIFIED
Subject Keywords:Gene regulation; Fluorescence; Confocal microscopy; Flow cytometry
Issue or Number:22
PubMed Central ID:PMC2587135
Record Number:CaltechAUTHORS:HSUbmcl08
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:HSUbmcl08
Official Citation:Carey F. Hsu, Peter B. Dervan, Quantitating the concentration of Py-Im polyamide–fluorescein conjugates in live cells, Bioorganic & Medicinal Chemistry Letters, Volume 18, Issue 22, 15 November 2008, Pages 5851-5855, ISSN 0960-894X, http://dx.doi.org/10.1016/j.bmcl.2008.05.063. (http://www.sciencedirect.com/science/article/pii/S0960894X0800560X)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:12671
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:19 Dec 2008 03:22
Last Modified:26 Nov 2019 11:15

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