Oxidative stress (OS) is associated with a wide variety of diseases and disorders. Detection of oxidative stress in living systems typically relies on fluorescent probes for reactive oxygen species (ROS), which is challenging because of their short life span and high reactivity. Oxidative damage caused by OS produces a more stable signal, but these biomarkers are usually detected using techniques that are not compatible with live cells. OS-induced biomolecule carbonylation is a stable modification that also possesses a chemically reactive functional group, and its detection typically employs a chemical reaction with a hydrazine-containing probe within the process. These hydrazone-forming reactions require strong acid catalysis or nucleophilic catalysis with an aromatic amine when performed on isolated biomaterial or on fixed cells. In live cells, however, hydrazone-forming reactions are surprisingly facile. Fluorophores possessing hydrazine or hydrazide functional groups can undergo reaction with carbonylated biomolecules in live cells, and these products can be observed using fluorescence microscopy. In this chapter, standard methods for detection of biomolecule carbonylation in cell lysate and in intact cells are enumerated. Protocols for fluorescently labeling biomolecule carbonylation in live cells are provided for commercially available fluorophores. Also described is a one-step protocol that employs one of the hydrazine-modified fluorophores developed in our lab, which are designed to be live-cell compatible and to undergo a spectral change upon hydrazone formation. Finally, a procedure for observing both biomolecule carbonylation and ROS production simultaneously is provided.
Keywords: Carbonylation; Fluorescence microscopy; Hydrazone; Live cells; Oxidative stress.
© 2020 Elsevier Inc. All rights reserved.