›› 2018, Vol. 38 ›› Issue (1): 10-.doi: 10.3969/j.issn.1674-8115.2018.01.003 [

• Original article (Basic research) • Previous Articles     Next Articles

Flow cytometry detection of cellular redox status with genetically encoded fluorescent probe

TIAN Ye, LIU Ao, DONG Rui, QI Xing, YI Jing, YANG Jie   

  1. Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
  • Online:2018-01-28 Published:2018-03-09
  • Supported by:
    National Natural Science Foundation of China, 31230037; Shanghai Natural
    Science Foundation, 16ZR1418400

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Abstract: Objective · To test the application of flow cytometry technique to detect the redox status with genetically encoded fluorescent probe roGFP2, to
compare it with laser scanning confocal microscope (LSCM), and to demonstrate the diversity of cellular redox status in HeLa and Panc-1 cell populations.
Methods · Time lapse imaging with LSCM was performed in single cell transfected with roGFP2 probe to detect the dynamic changes of 405 nm/488 nm
ratio (405/488 ratio). Flowcytometry technique was also performed in HeLa cells transfected with roGFP2 probe to detect the dynamic alterations of
405/488 ratio. The global cell population was analyzed and the subpopulations of different redox status were dissected. Flowcytometry technique was
further applied in Panc-1 cells with different CD24 or CD44 marker to detect the dynamic alterations of 405/488 ratio of roGFP2 and identify the different
redox status. Results · Time lapse imaging with LSCM showed that 405/488 ratio of roGFP2 dramatically changed in response to H2O2 in dose and time
dependent manner at a single cell level. When dozens of cells were chosen, the average ratio showed increased and dynamic trend. Compared to LSCM,
flow cytometry could detect the average of 405/488 ratio of roGFP2 as well. Meanwhile, with the application of flow cytometry the cell population can be
divided into three subpopulations based on 405/488 ratios, most in oxidized and medium redox status, few in reduced status. Using flow cytometry, CD24+/
CD44+ Panc-1 cells, pancreatic cancer stem cells, can be found to have overall lower 405/488 ratio and more percentage of subpopulation of reduced status under resting and stress condition. Conclusion · Flow cytometry technique can be applied to detect roGFP2 and has advantage in application to show the overall as well as diverse redox status in cell population. Flow cytometry detection of cellular redox status with genetically encoded probe can be a useful
tool in tumor cell biology and developmental biology research.

Key words: flow cytometry, redox status, genetically encoded fluorescent probe, roGFP2, tumor stem cells