Preparation of a cyanine-based fluorescent probe for highly selective detection of glutathione and its use in living cells and tissues of mice

Glutathione (GSH) is a major endogenous antioxidant that has a central role in cellular defense against toxins and free radicals. This protocol describes the preparation of CPCPDSASA, a cyanine-based near-infrared (NIR) fluorescent probe for the detection of GSH in cells and in vivo. CPCPDSASA is prepared with high yield through a simple two-step process. The first step is to react commercially available IR-780 iodide with excess anhydrous piperazine in anhydrous N, N-dimethyl formamide at 85 degrees C to form cyanine-piperazine (CPCP). The second step is the sulfonylation of CPCP with dansyl chloride in anhydrous dichloromethane. CPCPDSASA selectively detects GSH in cells, and it has been shown to not react with other biothiols such as cysteine (Cys) and homocysteine (Hcy). This probe can also be used to monitor the GSH level of mouse bone marrow-derived neutrophils (BMDNs). The preparation of probe CPCPDSASA takes 2 d, and experiments in cells and mice take 12-13 d.