Carcinogenesis, Teratogenesis & Mutagenesis ›› 2017, Vol. 29 ›› Issue (4): 284-288.doi: 10.3969/j.issn.1004-616x.2017.04.008

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Establishment of detection method for histone γ-H2AX phosphorylation based on flow cytometry

HUANG Pengcheng, ZHOU Changhui, LI Shenning, CHANG Yan   

  1. China State Institute of Pharmaceutical Industry, National Shanghai Center for New Drug Safety Evaluation & Research, Shanghai 201203, China
  • Received:2016-12-23 Revised:2017-04-13 Online:2017-07-31 Published:2017-07-31

Abstract:

OBJECTIVE: To established a method for automated detection of flow cytometry-based biomarker γ-H2AX,and to discussed the prospect of this method in drug screening for early genotoxicity. METHODS: The experiment was divided into two groups:-S9 group (long time treatment for 24 hours) and +S9 group (short time treatment for 4 hours). Then,4 suitable concentrations of Cyclophosphamide and Etoposide were selected using the CCK-8 assay. Treated TK6 cells were harvested according to standard operation procedures. After harvest,cells were double labeled with nucleic acid SYTOX Green and Alexa Fluor 647 conjugated anti-γ-H2AX antibody. BD Accuri C6 Flow cytometry was used to analyze the percentage of phosphorylated histone H2AX (γ-H2AX) cells. RESULTS: In the -S9 long-time treatment group,the ratio of γ-H2AX positive cells increased significantly compared with the solvent control group in different concentrations of Etoposide,and it showed a significant dose-dependent relationship. In the +S9 short-time treatment group,a significant increase in different concentrations of Cyclophosphamide was observed. CONCLUSION: Flow cytometry rapidly and accurately detected changes of γ-H2AX in cultured TK6 cells in vitro. Therefore,it is possible to detect γ-H2AX based on flow cytometry.

Key words: TK6 cell, γ-H2AX, flow cytometry, histone, phosphorylation

CLC Number: