Simple Fluorescence Assay for Cystine Uptake via the xCT in Cells Using Selenocystine and a Fluorescent Probe.

Takashi Shimomura, Norio Hirakawa, Yuya Ohuchi, Munetaka Ishiyama, Masanobu Shiga, Yuichiro Ueno

ACS sensors 2021 Jun 25

filter terms:

The cystine/glutamate antiporter (xCT) is a crucial transporter that maintains cellular redox balance by regulating intracellular glutathione synthesis via cystine uptake. However, no robust and simple method to determine the cystine uptake activity of xCT is currently available. We have developed a method to measure the xCT activity via the reaction of selenocysteine and fluorescein O,O'-diacrylate (FOdA). Selenocystine, a cystine analogue, is transported into cells through xCT on the cell membrane. The amount of the transported selenocystine was then determined by a reaction using tris(2-carboxyethyl)phosphine (TCEP) and FOdA in a weak acidic buffer at pH 6. Using this method, the cystine uptake activity of xCT in various cells and the inhibitory efficiency of xCT inhibitors, were evaluated.

Citation

Takashi Shimomura, Norio Hirakawa, Yuya Ohuchi, Munetaka Ishiyama, Masanobu Shiga, Yuichiro Ueno. Simple Fluorescence Assay for Cystine Uptake via the xCT in Cells Using Selenocystine and a Fluorescent Probe. ACS sensors. 2021 Jun 25;6(6):2125-2128