Abstract
Redox homeostasis is a fundamental property of living cells and responds actively to both cellular metabolism and external stimulus. The development of redox-sensitive GFP (roGFP) enables dynamic monitoring of changes in cellular redox poise (Hanson et al., 2014). When excited alternatively at 405 nm and 488 nm, these probes exhibit significant opposing shifts at the emission spectra (505-530 nm), which enables ratiometric measurement of relative redox values. A more oxidized environment results in a higher 405/488 ratio. Previously, successful application of roGFPs in leaf epidermis or root cells has been reported. Here we provide a protocol describing the application of roGFP1 imaging in growing pollen tubes by confocal laser scanning microscopy.
Keywords: Pollen tube, ROS, Redox level, Live cell imaging
Materials and Reagents
Equipment
Software
Procedure
The method consists of harvesting pollen grains, incubating them in liquid germination medium, performing live cell imaging and making ratiometric analysis. Pollen will start to germinate at about 30 min. Then ratiometric imaging using a confocal microscope is performed. This can be achieved in a few minutes. Ratiometric image analysis can be performed at a later time point.
Recipes
Acknowledgments
The roGFP1 was kindly provided by Dr. James Remington. This work was supported by the Natural Science Foundation of China (Grant 30970266 to Dong Zhang and Grant 31170291 to W.-H.T.) and the Ministry of Science and Technology of China (Grant 2012AA10A302 to W. -H. T.). We thank Xiao-Shu Gao for help with roGFP imaging and Yu-Jie Li for taking photographs.
References
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