Measuring adaptation dynamics to hydrogen peroxide in single human cells using fluorescent reporters

Abstract

ABSTRACTWe developed an experimental methodology to monitor response dynamics of single human cells to hydrogen peroxide. Our approach is based on fluidic control of both magnitude and time-evolution of the external perturbation, and on high-throughput imaging of intracellular fluorescent redox reporters. We applied step stimulus to MCF7 cells with hydrogen peroxide concentration in the range of 10 to 500μM. First, our data highlights dynamic adaptation of Reactive Oxygen Species (ROS) scavenging system at several time scales. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) level is rapidly restored within 10 min after a transient decrease, while glutathione (GSH) redox potential is slowly driven back toward pre-stimulus level (within one hour). Extra-cellular glucose is necessary for adaptation of both NADPH level and GSH system. Second, our results also reveal large cell-to-cell variability in the dynamic response to external ROS. Our experimental approach is readily usable to monitor other cellular redox systems such as thioredoxins. As response-heterogeneity can lead to fractional killing, we finally discuss how our methodology can be an opportunity to link dynamics of ROS scavenging and cellular fate such as cell death.