A novel method using confocal laser scanning microscopy for sensitive measurement of P-glycoprotein-mediated transport activity in Caco-2 cells
Objectives The aim of this study was to use time‐lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P‐glycoprotein activity in Caco‐2 cells. Methods The change in the fluorescence of residual rhodamine 123 at the apical and central regions of...
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| Published in: | Journal of pharmacy and pharmacology Vol. 63; no. 8; pp. 1015 - 1021 |
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| Main Authors: | , , , , , , , |
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Blackwell Publishing Ltd
01-08-2011
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| Abstract | Objectives The aim of this study was to use time‐lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P‐glycoprotein activity in Caco‐2 cells.
Methods The change in the fluorescence of residual rhodamine 123 at the apical and central regions of Caco‐2 cells was measured in the presence of digoxin or St John's wort by using time‐lapse confocal laser scanning microscopy. The data were compared with measurements made using conventional techniques, a fluorescence microplate reader and a fluorescence microscope.
Key findings The percentage decrease of rhodamine 123 caused by 10 µm digoxin or 0.1 µg/ml St John's wort was significantly larger in the apical region of the Caco‐2 cell than in the central region or in the whole cell. The digoxin‐induced inhibition in the apical region as measured by time‐lapse confocal laser scanning microscopy was greater than that measured in the whole cell by a microplate reader or a fluorescence microscope.
Conclusions The assay of residual rhodamine 123 in the apical region of Caco‐2 cells by confocal laser scanning microscopy was more sensitive than the conventional methods using a microplate reader or fluorescence microscopy. It will be a valuable screening tool for studying both the inhibition and induction of P‐glycoprotein activity. |
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| AbstractList | Objectives The aim of this study was to use time‐lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P‐glycoprotein activity in Caco‐2 cells.
Methods The change in the fluorescence of residual rhodamine 123 at the apical and central regions of Caco‐2 cells was measured in the presence of digoxin or St John's wort by using time‐lapse confocal laser scanning microscopy. The data were compared with measurements made using conventional techniques, a fluorescence microplate reader and a fluorescence microscope.
Key findings The percentage decrease of rhodamine 123 caused by 10 µm digoxin or 0.1 µg/ml St John's wort was significantly larger in the apical region of the Caco‐2 cell than in the central region or in the whole cell. The digoxin‐induced inhibition in the apical region as measured by time‐lapse confocal laser scanning microscopy was greater than that measured in the whole cell by a microplate reader or a fluorescence microscope.
Conclusions The assay of residual rhodamine 123 in the apical region of Caco‐2 cells by confocal laser scanning microscopy was more sensitive than the conventional methods using a microplate reader or fluorescence microscopy. It will be a valuable screening tool for studying both the inhibition and induction of P‐glycoprotein activity. The aim of this study was to use time-lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P-glycoprotein activity in Caco-2 cells. The change in the fluorescence of residual rhodamine 123 at the apical and central regions of Caco-2 cells was measured in the presence of digoxin or St John's wort by using time-lapse confocal laser scanning microscopy. The data were compared with measurements made using conventional techniques, a fluorescence microplate reader and a fluorescence microscope. The percentage decrease of rhodamine 123 caused by 10 µm digoxin or 0.1 µg/ml St John's wort was significantly larger in the apical region of the Caco-2 cell than in the central region or in the whole cell. The digoxin-induced inhibition in the apical region as measured by time-lapse confocal laser scanning microscopy was greater than that measured in the whole cell by a microplate reader or a fluorescence microscope. The assay of residual rhodamine 123 in the apical region of Caco-2 cells by confocal laser scanning microscopy was more sensitive than the conventional methods using a microplate reader or fluorescence microscopy. It will be a valuable screening tool for studying both the inhibition and induction of P-glycoprotein activity. OBJECTIVESThe aim of this study was to use time-lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P-glycoprotein activity in Caco-2 cells.METHODSThe change in the fluorescence of residual rhodamine 123 at the apical and central regions of Caco-2 cells was measured in the presence of digoxin or St John's wort by using time-lapse confocal laser scanning microscopy. The data were compared with measurements made using conventional techniques, a fluorescence microplate reader and a fluorescence microscope.KEY FINDINGSThe percentage decrease of rhodamine 123 caused by 10 µm digoxin or 0.1 µg/ml St John's wort was significantly larger in the apical region of the Caco-2 cell than in the central region or in the whole cell. The digoxin-induced inhibition in the apical region as measured by time-lapse confocal laser scanning microscopy was greater than that measured in the whole cell by a microplate reader or a fluorescence microscope.CONCLUSIONSThe assay of residual rhodamine 123 in the apical region of Caco-2 cells by confocal laser scanning microscopy was more sensitive than the conventional methods using a microplate reader or fluorescence microscopy. It will be a valuable screening tool for studying both the inhibition and induction of P-glycoprotein activity. |
| Author | Shinozuka, Kazumasa Fahmi, Odette A. Umegaki, Keizo Kagota, Satomi Nejime, Namie Wakuda, Hirokazu Tada, Yukari Yamada, Shizuo |
| Author_xml | – sequence: 1 givenname: Hirokazu surname: Wakuda fullname: Wakuda, Hirokazu organization: aDepartment of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya – sequence: 2 givenname: Namie surname: Nejime fullname: Nejime, Namie organization: aDepartment of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya – sequence: 3 givenname: Yukari surname: Tada fullname: Tada, Yukari organization: aDepartment of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya – sequence: 4 givenname: Satomi surname: Kagota fullname: Kagota, Satomi organization: aDepartment of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya – sequence: 5 givenname: Odette A. surname: Fahmi fullname: Fahmi, Odette A. organization: dPfizer Global Research & Development, Groton, CT, USA – sequence: 6 givenname: Keizo surname: Umegaki fullname: Umegaki, Keizo organization: cInformation Center, National Institute of Health and Nutrition, Tokyo, Japan and – sequence: 7 givenname: Shizuo surname: Yamada fullname: Yamada, Shizuo organization: bDepartment of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka – sequence: 8 givenname: Kazumasa surname: Shinozuka fullname: Shinozuka, Kazumasa email: kazumasa@mukogawa-u.ac.jp organization: aDepartment of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya |
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| Snippet | Objectives The aim of this study was to use time‐lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating... The aim of this study was to use time-lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P-glycoprotein... OBJECTIVESThe aim of this study was to use time-lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating... |
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| SubjectTerms | ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism Biological Transport - drug effects Biological Transport - physiology Caco-2 cells Caco-2 Cells - metabolism confocal laser scanning microscopy digoxin Digoxin - pharmacology Drug Interactions - physiology Fluorescent Dyes - metabolism Humans Hypericum Microscopy, Confocal - methods P-glycoprotein Plant Extracts - pharmacology Rhodamine 123 - metabolism St John's wort |
| Title | A novel method using confocal laser scanning microscopy for sensitive measurement of P-glycoprotein-mediated transport activity in Caco-2 cells |
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