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Sensors 2013, 13(12), 16330-16346; doi:10.3390/s131216330
Article

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

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Received: 18 October 2013; in revised form: 11 November 2013 / Accepted: 13 November 2013 / Published: 28 November 2013
(This article belongs to the Special Issue Fluorescent Biosensors)
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Abstract: To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity.
Keywords: mCerulean-mCitrine FRET-HIV protease sensor; ratiometric flow cytometry; sensitize emission FRET mCerulean-mCitrine FRET-HIV protease sensor; ratiometric flow cytometry; sensitize emission FRET
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Gaber, R.; Majerle, A.; Jerala, R.; Benčina, M. Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry. Sensors 2013, 13, 16330-16346.

AMA Style

Gaber R, Majerle A, Jerala R, Benčina M. Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry. Sensors. 2013; 13(12):16330-16346.

Chicago/Turabian Style

Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca. 2013. "Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry." Sensors 13, no. 12: 16330-16346.


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