Validation of an in vitro screen for phospholipidosis using a high-content biology platform
Identifieur interne : 001C31 ( Main/Exploration ); précédent : 001C30; suivant : 001C32Validation of an in vitro screen for phospholipidosis using a high-content biology platform
Auteurs : J. K. Morelli [États-Unis] ; M. Buehrle [États-Unis] ; F. Pognan [États-Unis] ; L. R. Barone [États-Unis] ; W. Fieles [États-Unis] ; P. J. Ciaccio [États-Unis]Source :
- Cell Biology and Toxicology [ 0742-2091 ] ; 2006-01-01.
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Abstract
Abstract: Several cationic amphiphilic drugs cause local or systemic phospholipidosis (PLD) after chronic exposure in preclinical species. PLD is characterized by the accumulation of drug, phospholipid, and concentric lamellar bodies in cellular lysosomes. We have developed a fluorescence-based in vitro screen that is predictive of PLD using the Cellomics ArrayScan high-content screening platform, which captures and analyzes images from 96-well cell culture microtiter plates using multichannel fluorescence microscopy. I-13.35 adherent mouse spleen macrophage cells were cultured with drug and a fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Drug concentrations were used in a range from 1 to $100 μmol/L. After 24 h incubations, the cells were fixed with formalin. NBD-PE uptake was quantified in controls and treated cells. Nuclei were identified by Hoechst 33258 staining and dead cells were identified using ethidium homodimer-2 incorporation. Thus, confounding accumulation of NBD-PE due to cytotoxicity that produces false-positive results at high concentrations was eliminated from quantitation by ethidium staining and employing cell gating (dead cell rejection). The assay was found to be both sensitive and selective in that 26 of 28 positive, phospholipidogenic controls and 8 of 8 negative, nonphospholipidogenic controls were correctly called.
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DOI: 10.1007/s10565-006-0176-z
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Ciaccio</name><affiliation wicri:level="1"><country xml:lang="fr">États-Unis</country><wicri:regionArea>Safety Assessment US</wicri:regionArea></affiliation></author></analytic><monogr></monogr><series><title level="j">Cell Biology and Toxicology</title><title level="j" type="abbrev">Cell Biol Toxicol</title><idno type="ISSN">0742-2091</idno><idno type="eISSN">1573-6822</idno><imprint><publisher>Kluwer Academic Publishers</publisher><pubPlace>Dordrecht</pubPlace><date type="published" when="2006-01-01">2006-01-01</date><biblScope unit="volume">22</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="15">15</biblScope><biblScope unit="page" to="27">27</biblScope></imprint><idno type="ISSN">0742-2091</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0742-2091</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>ArrayScan</term><term>I-13.35 cells</term><term>amiodarone</term><term>cationic amphiphilic drugs</term><term>high-content biology</term><term>phospholipidosis</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Several cationic amphiphilic drugs cause local or systemic phospholipidosis (PLD) after chronic exposure in preclinical species. PLD is characterized by the accumulation of drug, phospholipid, and concentric lamellar bodies in cellular lysosomes. We have developed a fluorescence-based in vitro screen that is predictive of PLD using the Cellomics ArrayScan high-content screening platform, which captures and analyzes images from 96-well cell culture microtiter plates using multichannel fluorescence microscopy. I-13.35 adherent mouse spleen macrophage cells were cultured with drug and a fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Drug concentrations were used in a range from 1 to $100 μmol/L. After 24 h incubations, the cells were fixed with formalin. NBD-PE uptake was quantified in controls and treated cells. Nuclei were identified by Hoechst 33258 staining and dead cells were identified using ethidium homodimer-2 incorporation. Thus, confounding accumulation of NBD-PE due to cytotoxicity that produces false-positive results at high concentrations was eliminated from quantitation by ethidium staining and employing cell gating (dead cell rejection). The assay was found to be both sensitive and selective in that 26 of 28 positive, phospholipidogenic controls and 8 of 8 negative, nonphospholipidogenic controls were correctly called.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Delaware</li></region></list><tree><country name="États-Unis"><noRegion><name sortKey="Morelli, J K" sort="Morelli, J K" uniqKey="Morelli J" first="J. K." last="Morelli">J. K. Morelli</name></noRegion><name sortKey="Barone, L R" sort="Barone, L R" uniqKey="Barone L" first="L. R." last="Barone">L. R. Barone</name><name sortKey="Buehrle, M" sort="Buehrle, M" uniqKey="Buehrle M" first="M." last="Buehrle">M. Buehrle</name><name sortKey="Ciaccio, P J" sort="Ciaccio, P J" uniqKey="Ciaccio P" first="P. 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