Many cellular events involve the alteration in redox equilibrium, globally or locally. In many cases, excessive reactive oxygen species (ROS) production is the underlying cause. Several green fluoresecence protein based indicators are constructed to measure redox status in cells, e.g, rxYFP and roGFPs, which allow real time detection. reduction and oxidization-sensitive GFP (RoGFPs) are more useful due to ratiometric variation by excitation, making the measurement more accurate. Utilizing one of those roGFPs called roGFP1, we establish a mitochondrial redox state probing platform in HeLa cells with laser scan confocal microscopy (LSCM) as detection system. Control experiments confirmed that our platform could produce stable ratiometric values, which made the data more accurately reflect the real environmental changes of redox status that roGFP1 probed. Using exogenous H₂O₂ and DTT, we evaluated the reactivity and reversibility of roGFP1. The minimal hydrogen peroxide concentration that roGFP1 could show detectable ratiometric changes in our system was about 200&mgr;M. Preliminarily applying our platform to exploring the redox status during apoptosis, we observed an increase in ratiometric, suggesting an excessive ROS production.

Date Published: 13 February 2007
PDF: 8 pages
Proc. SPIE 6438, Biophotonics and Immune Responses II, 64380K (13 February 2007); doi: 10.1117/12.701654

Published in SPIE Proceedings Vol. 6438:
Biophotonics and Immune Responses II
Wei R. Chen, Editor(s)