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| An improved high content screening method for compounds on activity of serotonin transporters |
| Hits 1410 Download times 1731 Received:January 28, 2016 |
| View Full Text View/Add Comment Download reader |
| DOI
10.11656/j.issn.1673-9043.2016.03.09 |
| Key Words
SERT;APP+;hSERT-HEK293 cell line;HCS |
| Author Name | Affiliation | E-mail | | LIU Yan-ting | Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Research and Development Center of Chinese Materia Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | | | YING Shu-song | Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Research and Development Center of Chinese Materia Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | | | WU Yi-jing | Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Research and Development Center of Chinese Materia Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | | | DONG Peng-zhi | Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Research and Development Center of Chinese Materia Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | | | WU Hong-hua | Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Research and Development Center of Chinese Materia Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | | | XU Yan-tong | Tianjin Key Laboratory of Modern Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Research and Development Center of Chinese Materia Medica, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China | tonyxu2015@sina.com |
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| Abstract
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| [Objective] To establish an improved high content screening (HCS) method for compounds on activity of serotonin transporter (SERT), a stably transfected human SERT cell line will be obtained and the test conditions of HCS method for SERT activity will be optimized. [Methods] To obtain the stably transfected SERT cell line, plasmid hSERT-pcDNA3 was transfected into the HEK293 cell line by polyethylenimine (PEI) and then the cells were screened by antibiotic G418. To verify the quality of this stably transfected cell line, expression of hSERT mRNA and protein was tested by Real-time PCR and Western blotting, respectively. To test the SERT reuptake activity, 4-(4-(Dimethylamino)phenyl)-1-methylpyridinium (APP+), a fluorescent substrate of SERT, was used and the test conditions of HCS assay were optimized. The positive drugs including selective serotonin reuptake inhibitor fluoxetine and enhancer tianeptine were used to confirm the reliability of this HCS assay. [Results] The stably tansfected hSERT-HEK293 cell line was obtained by PEI transfection and G418 screening. Compared to the HEK293 cells, expression of hSERT mRNA and protein in hSERT-HEK293 cells was significantly increased, and its transport ability of APP+ was also significantly increased, which could be specifically blocked by fluoxetine. The test conditions of HCS for SERT activity were optimized including the concentration of APP+, its incubation time and the method for data analysis. The SERT activity of hSERT-HEK293 cells were significantly inhibited by fluoxetine while significantly enhanced by tianeptine, which confirmed the reliability of this HCS method. [Conclusion] This study firstly optimized the HCS assay with APP+ in testing SERT activity, which obviously improved the sensitivity of HCS method for SERT activity, and it is the first time to show this HCS method suitable for screening SERT enhancers, so this study established an improved HCS method for compounds on hSERT activity. |
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