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[Application of high-content screening and flow cytometry analysis techniques on evaluation of myocardial fibroblasts proliferation.] [Article in Chinese]

CUI Wei, LI Yu-Lin^*, WU Yi-Na, ZHANG Cong-Cong, LIU Sa, ZHAO Li-Min

The Key laboratory of Remodeling Related Cardiovascular Diseases, Ministry of Education, Capital Medical University Affiliated Beijing Anzhen Hospital, Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing 100029, China

Abstract

The proliferation of cardiac fibroblasts (CFs) is a key pathological process in the cardiac remodeling. To establish an objective, quantitative method for the analysis of cell proliferation and cell cycle, we applied the high-content screening (HCS) and flow cytometry (FCM) techniques. CFs, isolated by enzyme digestion from newborn C57BL/6J mice, were serum starved for 12 h and then given 10% fetal bovine serum (FBS) for 24 h. Followed by BrdU and DAPI (or 7-AAD) staining, CFs proliferation and cell cycle were analyzed by HCS and FCM, respectively. Discoidin domain receptor 2 (DDR2) staining indicated that the purity of isolated CFs was over 95%. (1) HCS analysis showed that the ratio of BrdU-positive cells was significantly increased in 10% FBS treated group compared with that in serum-free control group [(12.96 ± 0.67)% vs (2.77 ± 0.33)%; P < 0.05]. Cell cycle analysis showed that CFs in G0/G1 phase were diploid, and CFs in S phase were companied with proliferation, DNA replication and enlarged nuclei; CFs in G2 phase were tetraploid, and CFs in M phase produced two identical cells (2N). (2) FCM analysis showed that the ratio of BrdU-positive cells was increased in 10% FBS treated group compared with that in the control group [(11.10 ± 0.42)% vs (2.22 ± 0.31)%; P < 0.05]; DNA content histogram of cell cycle analysis indicated that the platform of S phase elevated in 10% FBS group compared with control group. (3) There were no differences between the two methods in the results of proliferation and cell cycle analysis. In conclusion, HCS and FCM methods are reliable, stable and consistent in assessment of the proliferation and cell cycle in CFs.

Key words: high content; flow cytometry; fibroblasts; cell proliferation; cell cycle

Received: 2013-09-30　　Accepted: 2013-12-24

Corresponding author: 李玉琳　　E-mail: lyllyl_1111@163.com

Citing This Article：

CUI Wei, LI Yu-Lin, WU Yi-Na, ZHANG Cong-Cong, LIU Sa, ZHAO Li-Min. [Application of high-content screening and flow cytometry analysis techniques on evaluation of myocardial fibroblasts proliferation.] [Article in Chinese]. Acta Physiol Sin 2014; 66 (2): 215-222 (in Chinese with English abstract).