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Primary Culture of Mouse Proximal Tubular Cells and Characterization of Cytochrome P450 Enzyme Activities
Bo Hai1, Zhao-Hui Chen1, Zhi Li, Lu-Lu Li, Chuan-Guo Xiao*
Department of Urology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
Abstract: The renal tubular segments were collected after I collagenase digestion and mesh filtration， followed by Percoll density centrifugation. The cell types were identified by immunocytochemistry using anti-cytokeratin 18. Cytochrome P450 (CYPIA1) activity was characterized using 7-ethoxyresorufin-O-deethylase (EROD) method at day 0， 1， 3 and 7 after isolation. Flow cytometry showed the purity of proximal tubular cell (PTC) reached 98% in secondary cells， which proved the methods of mesh filtration and Percoll density centrifugation were effective. A relatively high CYPIA1 activity could be observed in the first 24 hours after isolation， and then the substrates were metabolized at significant lower rates in 3 and 7 days.
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