The production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress
are tightly linked. The generation of ROS can be both the cause and a consequence
of ER stress pathways, and an increasing number of human diseases are characterized
by tissue atrophy in response to ER stress and oxidative injury. For the assessment
of modulators of ER luminal ROS generation and for mechanistic studies, methods to
monitor changes in ER reduction-oxidation (redox) states in a time-resolved and organelle-specific
manner are needed. This has been greatly facilitated by the development of genetically
encoded fluorescent probes, which can be targeted to different subcellular locations
by specific amino acid extensions. One of these probes is the yellow fluorescent protein-based
redox biosensor, HyPer. Here, we provide a protocol for the time-resolved monitoring
of the oxidizing milieu in the ER of adherent mammalian cells using the ratiometric
sensor, HyPerER, which is specifically targeted to the ER lumen.
