P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods
Identifieur interne : 000067 ( Pmc/Checkpoint ); précédent : 000066; suivant : 000068P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods
Auteurs : Jennifer Pasquier [France, États-Unis, Qatar] ; Damien Rioult [France] ; Nadine Abu-Kaoud [Qatar] ; Sabine Marie [France] ; Arash Rafii [États-Unis, Qatar] ; Bella S. Guerrouahen [États-Unis, Qatar] ; Frank Le Foll [France]Source :
- BioMed Research International [ 2314-6133 ] ; 2013.
Abstract
Url:
DOI: 10.1155/2013/676845
PubMed: 24350282
PubMed Central: 3848087
Affiliations:
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods</title><author><name sortKey="Pasquier, Jennifer" sort="Pasquier, Jennifer" uniqKey="Pasquier J" first="Jennifer" last="Pasquier">Jennifer Pasquier</name><affiliation wicri:level="3"><nlm:aff id="I1">Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex</wicri:regionArea><placeName><region type="region" nuts="2">Région Normandie</region><region type="old region" nuts="2">Haute-Normandie</region><settlement type="city">Le Havre</settlement></placeName></affiliation><affiliation wicri:level="2"><nlm:aff id="I2">Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="I3">Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144, Qatar</nlm:aff><country xml:lang="fr">Qatar</country><wicri:regionArea>Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144</wicri:regionArea><wicri:noRegion>Doha 24144</wicri:noRegion></affiliation></author><author><name sortKey="Rioult, Damien" sort="Rioult, Damien" uniqKey="Rioult D" first="Damien" last="Rioult">Damien Rioult</name><affiliation wicri:level="3"><nlm:aff id="I1">Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex</wicri:regionArea><placeName><region type="region" nuts="2">Région Normandie</region><region type="old region" nuts="2">Haute-Normandie</region><settlement type="city">Le Havre</settlement></placeName></affiliation></author><author><name sortKey="Abu Kaoud, Nadine" sort="Abu Kaoud, Nadine" uniqKey="Abu Kaoud N" first="Nadine" last="Abu-Kaoud">Nadine Abu-Kaoud</name><affiliation wicri:level="1"><nlm:aff id="I3">Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144, Qatar</nlm:aff><country xml:lang="fr">Qatar</country><wicri:regionArea>Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144</wicri:regionArea><wicri:noRegion>Doha 24144</wicri:noRegion></affiliation></author><author><name sortKey="Marie, Sabine" sort="Marie, Sabine" uniqKey="Marie S" first="Sabine" last="Marie">Sabine Marie</name><affiliation wicri:level="3"><nlm:aff id="I1">Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex</wicri:regionArea><placeName><region type="region" nuts="2">Région Normandie</region><region type="old region" nuts="2">Haute-Normandie</region><settlement type="city">Le Havre</settlement></placeName></affiliation></author><author><name sortKey="Rafii, Arash" sort="Rafii, Arash" uniqKey="Rafii A" first="Arash" last="Rafii">Arash Rafii</name><affiliation wicri:level="2"><nlm:aff id="I2">Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="I3">Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144, Qatar</nlm:aff><country xml:lang="fr">Qatar</country><wicri:regionArea>Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144</wicri:regionArea><wicri:noRegion>Doha 24144</wicri:noRegion></affiliation></author><author><name sortKey="Guerrouahen, Bella S" sort="Guerrouahen, Bella S" uniqKey="Guerrouahen B" first="Bella S." last="Guerrouahen">Bella S. Guerrouahen</name><affiliation wicri:level="2"><nlm:aff id="I2">Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="I3">Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144, Qatar</nlm:aff><country xml:lang="fr">Qatar</country><wicri:regionArea>Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144</wicri:regionArea><wicri:noRegion>Doha 24144</wicri:noRegion></affiliation></author><author><name sortKey="Le Foll, Frank" sort="Le Foll, Frank" uniqKey="Le Foll F" first="Frank" last="Le Foll">Frank Le Foll</name><affiliation wicri:level="3"><nlm:aff id="I1">Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex</wicri:regionArea><placeName><region type="region" nuts="2">Région Normandie</region><region type="old region" nuts="2">Haute-Normandie</region><settlement type="city">Le Havre</settlement></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24350282</idno><idno type="pmc">3848087</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848087</idno><idno type="RBID">PMC:3848087</idno><idno type="doi">10.1155/2013/676845</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">000208</idno><idno type="wicri:Area/Pmc/Curation">000206</idno><idno type="wicri:Area/Pmc/Checkpoint">000067</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods</title><author><name sortKey="Pasquier, Jennifer" sort="Pasquier, Jennifer" uniqKey="Pasquier J" first="Jennifer" last="Pasquier">Jennifer Pasquier</name><affiliation wicri:level="3"><nlm:aff id="I1">Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex</wicri:regionArea><placeName><region type="region" nuts="2">Région Normandie</region><region type="old region" nuts="2">Haute-Normandie</region><settlement type="city">Le Havre</settlement></placeName></affiliation><affiliation wicri:level="2"><nlm:aff id="I2">Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="I3">Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144, Qatar</nlm:aff><country xml:lang="fr">Qatar</country><wicri:regionArea>Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144</wicri:regionArea><wicri:noRegion>Doha 24144</wicri:noRegion></affiliation></author><author><name sortKey="Rioult, Damien" sort="Rioult, Damien" uniqKey="Rioult D" first="Damien" last="Rioult">Damien Rioult</name><affiliation wicri:level="3"><nlm:aff id="I1">Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex</wicri:regionArea><placeName><region type="region" nuts="2">Région Normandie</region><region type="old region" nuts="2">Haute-Normandie</region><settlement type="city">Le Havre</settlement></placeName></affiliation></author><author><name sortKey="Abu Kaoud, Nadine" sort="Abu Kaoud, Nadine" uniqKey="Abu Kaoud N" first="Nadine" last="Abu-Kaoud">Nadine Abu-Kaoud</name><affiliation wicri:level="1"><nlm:aff id="I3">Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144, Qatar</nlm:aff><country xml:lang="fr">Qatar</country><wicri:regionArea>Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144</wicri:regionArea><wicri:noRegion>Doha 24144</wicri:noRegion></affiliation></author><author><name sortKey="Marie, Sabine" sort="Marie, Sabine" uniqKey="Marie S" first="Sabine" last="Marie">Sabine Marie</name><affiliation wicri:level="3"><nlm:aff id="I1">Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex</wicri:regionArea><placeName><region type="region" nuts="2">Région Normandie</region><region type="old region" nuts="2">Haute-Normandie</region><settlement type="city">Le Havre</settlement></placeName></affiliation></author><author><name sortKey="Rafii, Arash" sort="Rafii, Arash" uniqKey="Rafii A" first="Arash" last="Rafii">Arash Rafii</name><affiliation wicri:level="2"><nlm:aff id="I2">Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="I3">Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144, Qatar</nlm:aff><country xml:lang="fr">Qatar</country><wicri:regionArea>Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144</wicri:regionArea><wicri:noRegion>Doha 24144</wicri:noRegion></affiliation></author><author><name sortKey="Guerrouahen, Bella S" sort="Guerrouahen, Bella S" uniqKey="Guerrouahen B" first="Bella S." last="Guerrouahen">Bella S. Guerrouahen</name><affiliation wicri:level="2"><nlm:aff id="I2">Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="I3">Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144, Qatar</nlm:aff><country xml:lang="fr">Qatar</country><wicri:regionArea>Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144</wicri:regionArea><wicri:noRegion>Doha 24144</wicri:noRegion></affiliation></author><author><name sortKey="Le Foll, Frank" sort="Le Foll, Frank" uniqKey="Le Foll F" first="Frank" last="Le Foll">Frank Le Foll</name><affiliation wicri:level="3"><nlm:aff id="I1">Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex</wicri:regionArea><placeName><region type="region" nuts="2">Région Normandie</region><region type="old region" nuts="2">Haute-Normandie</region><settlement type="city">Le Havre</settlement></placeName></affiliation></author></analytic><series><title level="j">BioMed Research International</title><idno type="ISSN">2314-6133</idno><idno type="eISSN">2314-6141</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Background</italic>. P-gp expression has been linked to the efflux of chemotherapeutic drugs in human cancers leading to multidrug resistance. Fluorescence techniques have been widely applied to measure the P-gp activity. In this paper, there is a comparison between the advantages of two fluorescence approaches of commonly available and affordable instruments: the microplate reader (MPR) and the flow cytometer to detect the P-gp efflux activity using calcein-AM. <italic>Results</italic>. The selectivity, sensibility, and reproducibility of the two methods have been defined. Our results showed that the MPR is more powerful for the detection of small inhibition, whereas the flow cytometry method is more reliable at higher concentrations of the inhibitors. We showed that to determine precisely the inhibition efficacy the flow cytometry is better; hence, to get the correct <italic>E</italic><sub>max</sub> and EC<sub>50</sub> values, we cannot only rely on the MPR. <italic>Conclusion</italic>. Both techniques can potentially be used extensively in the pharmaceutical industry for high-throughput drug screening and in biology laboratories for academic research, monitoring the P-gp efflux in specific assays.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Pluen, A" uniqKey="Pluen A">A Pluen</name></author><author><name sortKey="Boucher, Y" uniqKey="Boucher Y">Y Boucher</name></author><author><name sortKey="Ramanujan, S" uniqKey="Ramanujan S">S Ramanujan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Druker, Bj" uniqKey="Druker B">BJ Druker</name></author><author><name sortKey="Sawyers, Cl" uniqKey="Sawyers C">CL Sawyers</name></author><author><name sortKey="Kantarjian, H" uniqKey="Kantarjian H">H Kantarjian</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Biedler, Jl" uniqKey="Biedler J">JL Biedler</name></author><author><name sortKey="Riehm, H" uniqKey="Riehm H">H Riehm</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Endicott, Ja" uniqKey="Endicott J">JA Endicott</name></author><author><name sortKey="Ling, V" uniqKey="Ling V">V Ling</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bosch, I" uniqKey="Bosch I">I Bosch</name></author><author><name sortKey="Croop, J" uniqKey="Croop J">J Croop</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ambudkar, Sv" uniqKey="Ambudkar S">SV Ambudkar</name></author><author><name sortKey="Dey, S" uniqKey="Dey S">S Dey</name></author><author><name sortKey="Hrycyna, Ca" uniqKey="Hrycyna C">CA Hrycyna</name></author><author><name sortKey="Ramachandra, M" uniqKey="Ramachandra M">M Ramachandra</name></author><author><name sortKey="Pastan, I" uniqKey="Pastan I">I Pastan</name></author><author><name sortKey="Gottesman, Mm" uniqKey="Gottesman M">MM Gottesman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ambudkar, Sv" uniqKey="Ambudkar S">SV Ambudkar</name></author><author><name sortKey="Kimchi Sarfaty, C" uniqKey="Kimchi Sarfaty C">C Kimchi-Sarfaty</name></author><author><name sortKey="Sauna, Ze" uniqKey="Sauna Z">ZE Sauna</name></author><author><name sortKey="Gottesman, Mm" uniqKey="Gottesman M">MM Gottesman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schinkel, Ah" uniqKey="Schinkel A">AH Schinkel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Holland, Ib" uniqKey="Holland I">IB Holland</name></author><author><name sortKey="A Blight, M" uniqKey="A Blight M">M A. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Biomed Res Int</journal-id><journal-id journal-id-type="iso-abbrev">Biomed Res Int</journal-id><journal-id journal-id-type="publisher-id">BMRI</journal-id><journal-title-group><journal-title>BioMed Research International</journal-title></journal-title-group><issn pub-type="ppub">2314-6133</issn><issn pub-type="epub">2314-6141</issn><publisher><publisher-name>Hindawi Publishing Corporation</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24350282</article-id><article-id pub-id-type="pmc">3848087</article-id><article-id pub-id-type="doi">10.1155/2013/676845</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Pasquier</surname><given-names>Jennifer</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="aff" rid="I2"><sup>2</sup></xref><xref ref-type="aff" rid="I3"><sup>3</sup></xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Rioult</surname><given-names>Damien</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Abu-Kaoud</surname><given-names>Nadine</given-names></name><xref ref-type="aff" rid="I3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Marie</surname><given-names>Sabine</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Rafii</surname><given-names>Arash</given-names></name><xref ref-type="aff" rid="I2"><sup>2</sup></xref><xref ref-type="aff" rid="I3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Guerrouahen</surname><given-names>Bella S.</given-names></name><xref ref-type="aff" rid="I2"><sup>2</sup></xref><xref ref-type="aff" rid="I3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Le Foll</surname><given-names>Frank</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib></contrib-group><aff id="I1"><sup>1</sup>Laboratory of Ecotoxicology UPRES EA 3222, IFRMP 23, University of Le Havre, 76058 Le Havre Cedex, France</aff><aff id="I2"><sup>2</sup>Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10022, USA</aff><aff id="I3"><sup>3</sup>Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Doha 24144, Qatar</aff><author-notes><corresp id="cor1">*Jennifer Pasquier: <email>jep2026@qatar-med.cornell.edu</email></corresp><fn fn-type="other"><p>Academic Editor: Beric Henderson</p></fn></author-notes><pub-date pub-type="ppub"><year>2013</year></pub-date><pub-date pub-type="epub"><day>17</day><month>11</month><year>2013</year></pub-date><volume>2013</volume><elocation-id>676845</elocation-id><history><date date-type="received"><day>17</day><month>6</month><year>2013</year></date><date date-type="accepted"><day>29</day><month>9</month><year>2013</year></date></history><permissions><copyright-statement>Copyright © 2013 Jennifer Pasquier et al.</copyright-statement><copyright-year>2013</copyright-year><license license-type="open-access"><license-p>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.</license-p></license></permissions><abstract><p><italic>Background</italic>. P-gp expression has been linked to the efflux of chemotherapeutic drugs in human cancers leading to multidrug resistance. Fluorescence techniques have been widely applied to measure the P-gp activity. In this paper, there is a comparison between the advantages of two fluorescence approaches of commonly available and affordable instruments: the microplate reader (MPR) and the flow cytometer to detect the P-gp efflux activity using calcein-AM. <italic>Results</italic>. The selectivity, sensibility, and reproducibility of the two methods have been defined. Our results showed that the MPR is more powerful for the detection of small inhibition, whereas the flow cytometry method is more reliable at higher concentrations of the inhibitors. We showed that to determine precisely the inhibition efficacy the flow cytometry is better; hence, to get the correct <italic>E</italic><sub>max</sub> and EC<sub>50</sub> values, we cannot only rely on the MPR. <italic>Conclusion</italic>. Both techniques can potentially be used extensively in the pharmaceutical industry for high-throughput drug screening and in biology laboratories for academic research, monitoring the P-gp efflux in specific assays.</p></abstract></article-meta></front><floats-group><fig id="fig1" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Effects of P-glycoprotein blockers on calcein-AM efflux obtained by quantifying whole well or cell-normalized well fluorescence with a microplate reader. P-gp activity was measured as the ability to efflux the fluorescent P-gp allocrite calcein-AM. MCF-7/Doxo (a) or Hs578T/Doxo (b) multidrug resistant P-gp overexpressing breast cancer cell lines were incubated with 0, 1, or 10 <italic>μ</italic>M of the P-gp antagonists verapamil (VRP) or PSC833. Left: global cell population P-gp activity was quantified by measuring whole well fluorescence (arbitrary units, A.U.) in 6-plicates for 10 repeated experiments in each condition. Middle: a normalized single cell P-gp activity (A.U.) was obtained from cell population in each well by computing the ratio of whole well fluorescence to the number of cells in the corresponding well. In this respect, an MTT assay was systematically performed after the fluorescence readings to determine the number of cells in each well of the microplate. Right: MTT calibration curves. MTT optical density (MTT OD) varied as a function of the number of cells (NC) deposited in the wells, in a saturation type Mickaelis-Menten relationship. For calibration, the number of cells was determined by using a Malassez counting chamber. Curve fitting to the data gave the following parameters: <italic>a</italic> = 5,27; <italic>b</italic> = 175092 for MCF-7/Doxo cells and <italic>a</italic> = 7,64; <italic>b</italic> = 113025 for Hs578T/Doxo cells, respectively. Data are presented as mean ± sem with <italic>n</italic> = 10 independent assays per data point. Results significantly different from the control are indicated (*<italic>P</italic> < 0,05; **<italic>P</italic> < 0,01; ***<italic>P</italic> < 0,001; paired Student's <italic>t</italic>-test).</p></caption><graphic xlink:href="BMRI2013-676845.001"></graphic></fig><fig id="fig2" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Effects of P-glycoprotein blockers on calcein-AM efflux obtained by analyzing single cell fluorescence (FL1) or fluorescence concentration (FL1-FC) with a flow cytometer. P-gp activity was followed with calcein-AM as a fluorescent probe. In each flow cytometry measurement, a sample of 10 000 cells was analyzed. (a) Top panels, left: superimposed all-events histograms of calcein fluorescence distribution (log scale) in control MCF-7/Doxo (solid gray histogram) and MCF-7/Doxo preincubated with the P-gp noncompetitive antagonist PSC-833 (10 <italic>μ</italic>M, open histogram). Right: the amount of fluorescence per cell is expressed as FL1-FC (fluorescent light in channel 1-fluorescence concentration) which is the fluorescent light (FL) divided by the electronic volume (EV) determined by the flow cytometer according to the Coulter Principle. The EV distribution of the sample is given in insert. Bottom panels: the two histograms present the mean fluorescence, FL1 (left) and the cell volume normalized fluorescence, FL1-FC (right) without or with 1 <italic>μ</italic>M or 10 <italic>μ</italic>M of PSC833 or verapamil (VRP) for 10 repeated experiments. (b) The same experiments were carried out in Hs578T/Doxo. Data are presented as mean ± sem with <italic>n</italic> = 10 independent assays per data point. *<italic>P</italic> < 0,05, **<italic>P</italic> < 0,01, ***<italic>P</italic> < 0,001. </p></caption><graphic xlink:href="BMRI2013-676845.002"></graphic></fig><fig id="fig3" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Microplate reader and flow cytometer fluorescence responses linearity. MCF-7 drug-sensitive parental cells were incubated during 15 minutes in the dark with 0,05; 0,1; 0,25; 0,5; 0,75, or 1 <italic>μ</italic>M of calcein-AM. Cells were analysed by flow cytometry for mean volume-normalized fluorescence (FL1-FC, (a)) and by a microplate reader for cell-normalized well fluorescence (b). Data are presented as mean ± sem with <italic>n</italic> = 10 independent assays per data point.</p></caption><graphic xlink:href="BMRI2013-676845.003"></graphic></fig><fig id="fig4" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Intramethod technical replicability within ten independent biological samples. (a) Graphs show coefficient of variation (CV) of ten independent samples analyzed by flow cytometry after treatment with various concentrations of VRP (<italic>left</italic>) or <italic>PSC833 (right</italic>). A sample consists in the analysis of 10 000 cells. CV is expressed as the ratio of the standard deviation (SD) of the peak to the mean channel value (in percent) and provides a measure of the variability in signal intensity. (b) Coefficient of variation (CV = 100 × SD/mean) of 6-plicated measurements of fluorescence obtained with a microplate reader for ten independent biological samples exposed to different concentrations of VRP.</p></caption><graphic xlink:href="BMRI2013-676845.004"></graphic></fig><fig id="fig5" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Intermethods comparison of repeatability for increasing PSC833 concentration. Each graph shows the distribution of calcein fluorescence in 10 samples analyzed with both methods. Cell-normalized well fluorescence in microplate reader is expressed as a function of cell fluorescence concentration (FL1-FC) in flow cytometry for increasing concentrations of PSC833. Straight line corresponds to the mean methods fluorescence ratio.</p></caption><graphic xlink:href="BMRI2013-676845.005"></graphic></fig><fig id="fig6" orientation="portrait" position="float"><label>Figure 6</label><caption><p>Method responses differential as a function of mean fluorescence. The graph shows the differences between fluorescence measured in flow cytometry (FL1-FC) and microplate reader (cell-normalized well fluorescence) as function of mean fluorescence measured in these two methods. Gap between flow cytometry and microplate reader responses linearly increased with P-gp blockers-induced fluorescence accumulation.</p></caption><graphic xlink:href="BMRI2013-676845.006"></graphic></fig><fig id="fig7" orientation="portrait" position="float"><label>Figure 7</label><caption><p>Intermethods comparison of PSC833 and verapamil doses responses curves expressed as mean fluorescence. Superimposed microplate reader and flow cytometry semilogarithmic doses responses curves showing the effect of increasing verapamil or PSC833 concentrations on calcein accumulation in MCF-7/Doxo (a) or Hs578T (b). Each point represents means ± sem (10 independent experiments) of cell-normalized well fluorescence (open circles) or flow cytometry mean FL1-FC (filled circles) expressed as the ratio of signals in the presence of blocker to signals in control conditions. Ligand binding sigmoidal doses responses curves were fitted to the data to obtain blockers potencies (half-maximal effective concentration, EC<sub>50</sub>) and efficacies (maximum response, <italic>E</italic><sub>max</sub>).</p></caption><graphic xlink:href="BMRI2013-676845.007"></graphic></fig><fig id="fig8" orientation="portrait" position="float"><label>Figure 8</label><caption><p>Flow cytometry PSC833 doses responses curves obtained by analyzing modal single cell fluorescence concentration (FL1-FC). Semilogarithmic doses responses curves showing the effect of increasing PSC833 concentrations on calcein accumulation in MCF-7/Doxo. Each point represents peak (modal) ± sem (10 independent experiments) of FL1-FC in MCF7/Doxo (a) or Hs578T/Doxo (b) expressed as the ratio of signal in the presence of PSC833 to signal in control conditions. Ligand binding sigmoidal doses responses curves were fitted to the data to obtain PSC833 potency (half-maximal effective concentration, EC<sub>50</sub>) and efficacy (maximum response, <italic>E</italic><sub>max</sub>).</p></caption><graphic xlink:href="BMRI2013-676845.008"></graphic></fig><table-wrap id="tab1" orientation="portrait" position="float"><label>Table 1</label><caption><p>Intermethods comparison of fluorescence increase detection thresholds.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"> </th><th align="center" colspan="2" rowspan="1">MCF-7</th><th align="center" colspan="2" rowspan="1">Hs578T</th></tr><tr><th align="left" rowspan="1" colspan="1"> </th><th align="center" rowspan="1" colspan="1">0,1 <italic>µ</italic>M VRP</th><th align="center" rowspan="1" colspan="1">0,1 <italic>µ</italic>M PSC833</th><th align="center" rowspan="1" colspan="1">0,1 <italic>µ</italic>M VRP</th><th align="center" rowspan="1" colspan="1">0,1 <italic>µ</italic>M PSC833</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>Flow cytometer </italic></td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr><td align="left" rowspan="1" colspan="1">Mean FL1</td><td align="center" rowspan="1" colspan="1">∗∗</td><td align="center" rowspan="1" colspan="1">∗∗∗</td><td align="center" rowspan="1" colspan="1">∗</td><td align="center" rowspan="1" colspan="1">∗∗∗</td></tr><tr><td align="left" rowspan="1" colspan="1">Mean FL1-FC</td><td align="center" rowspan="1" colspan="1">∗∗</td><td align="center" rowspan="1" colspan="1">∗∗∗</td><td align="center" rowspan="1" colspan="1">∗</td><td align="center" rowspan="1" colspan="1">∗∗</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Microplate reader </italic></td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr><td align="left" rowspan="1" colspan="1">Well fluorescence</td><td align="center" rowspan="1" colspan="1">∗∗∗</td><td align="center" rowspan="1" colspan="1">∗</td><td align="center" rowspan="1" colspan="1">∗∗</td><td align="center" rowspan="1" colspan="1">∗∗</td></tr><tr><td align="left" rowspan="1" colspan="1">Single cell fluorescence</td><td align="center" rowspan="1" colspan="1">∗∗∗</td><td align="center" rowspan="1" colspan="1">∗∗∗</td><td align="center" rowspan="1" colspan="1">∗∗</td><td align="center" rowspan="1" colspan="1">∗∗</td></tr></tbody></table><table-wrap-foot><fn><p>Flow cytometer (mean FL1 and mean FL1-FC) and microplate reader (whole well fluorescence and mean single cell fluorescence) signal increase detection thresholds were estimated by testing groups of untreated cells and cells treated with the lowest P-gp antagonists concentration (0,1 <italic>µ</italic>M) for significant differences (*<italic>P</italic> < 0,05; **<italic>P</italic> < 0,01; ***<italic>P</italic> < 0,001; paired Student's <italic>t</italic>-test; <italic>n</italic> = 10).</p></fn></table-wrap-foot></table-wrap><table-wrap id="tab2" orientation="portrait" position="float"><label>Table 2</label><caption><p>EC<sub>50</sub> and <italic>E</italic><sub>max</sub> values obtained from the doses responses curves. </p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1"> </th><th align="center" colspan="2" rowspan="1">EC<sub>50</sub> (<italic>µ</italic>M)</th><th align="center" colspan="2" rowspan="1"><italic>E</italic><sub>max</sub></th></tr><tr><th align="center" rowspan="1" colspan="1">FC</th><th align="center" rowspan="1" colspan="1">MPR</th><th align="center" rowspan="1" colspan="1">FC</th><th align="center" rowspan="1" colspan="1">MPR</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">MCF-7/Doxo <break></break>PSC833</td><td align="center" rowspan="1" colspan="1">1,40</td><td align="center" rowspan="1" colspan="1">1,23</td><td align="center" rowspan="1" colspan="1">16,03</td><td align="center" rowspan="1" colspan="1">6,01</td></tr><tr><td align="left" rowspan="1" colspan="1">MCF-7/Doxo <break></break>VRP</td><td align="center" rowspan="1" colspan="1">28,29</td><td align="center" rowspan="1" colspan="1">68,04</td><td align="center" rowspan="1" colspan="1">24,59</td><td align="center" rowspan="1" colspan="1">10,39</td></tr><tr><td align="left" rowspan="1" colspan="1">Hs578T/Doxo <break></break>PSC833</td><td align="center" rowspan="1" colspan="1">1,83</td><td align="center" rowspan="1" colspan="1">1,40</td><td align="center" rowspan="1" colspan="1">11,99</td><td align="center" rowspan="1" colspan="1">4,48</td></tr><tr><td align="left" rowspan="1" colspan="1">Hs578T/Doxo<break></break>VRP</td><td align="center" rowspan="1" colspan="1">102,00</td><td align="center" rowspan="1" colspan="1">24,96</td><td align="center" rowspan="1" colspan="1">44,70</td><td align="center" rowspan="1" colspan="1">6,27</td></tr></tbody></table><table-wrap-foot><fn><p>Values are given for the two methods (flow cytometry—FC and microplate reader—MPR) with the two different antagonists for the two cells lines, MCF-7/Doxo and Hs578T.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc><affiliations><list><country><li>France</li><li>Qatar</li><li>États-Unis</li></country><region><li>Haute-Normandie</li><li>Région Normandie</li><li>État de New York</li></region><settlement><li>Le Havre</li></settlement></list><tree><country name="France"><region name="Région Normandie"><name sortKey="Pasquier, Jennifer" sort="Pasquier, Jennifer" uniqKey="Pasquier J" first="Jennifer" last="Pasquier">Jennifer Pasquier</name></region><name sortKey="Le Foll, Frank" sort="Le Foll, Frank" uniqKey="Le Foll F" first="Frank" last="Le Foll">Frank Le Foll</name><name sortKey="Marie, Sabine" sort="Marie, Sabine" uniqKey="Marie S" first="Sabine" last="Marie">Sabine Marie</name><name sortKey="Rioult, Damien" sort="Rioult, Damien" uniqKey="Rioult D" first="Damien" last="Rioult">Damien Rioult</name></country><country name="États-Unis"><region name="État de New York"><name sortKey="Pasquier, Jennifer" sort="Pasquier, Jennifer" uniqKey="Pasquier J" first="Jennifer" last="Pasquier">Jennifer Pasquier</name></region><name sortKey="Guerrouahen, Bella S" sort="Guerrouahen, Bella S" uniqKey="Guerrouahen B" first="Bella S." last="Guerrouahen">Bella S. Guerrouahen</name><name sortKey="Rafii, Arash" sort="Rafii, Arash" uniqKey="Rafii A" first="Arash" last="Rafii">Arash Rafii</name></country><country name="Qatar"><noRegion><name sortKey="Pasquier, Jennifer" sort="Pasquier, Jennifer" uniqKey="Pasquier J" first="Jennifer" last="Pasquier">Jennifer Pasquier</name></noRegion><name sortKey="Abu Kaoud, Nadine" sort="Abu Kaoud, Nadine" uniqKey="Abu Kaoud N" first="Nadine" last="Abu-Kaoud">Nadine Abu-Kaoud</name><name sortKey="Guerrouahen, Bella S" sort="Guerrouahen, Bella S" uniqKey="Guerrouahen B" first="Bella S." last="Guerrouahen">Bella S. Guerrouahen</name><name sortKey="Rafii, Arash" sort="Rafii, Arash" uniqKey="Rafii A" first="Arash" last="Rafii">Arash Rafii</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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