Principle

The Cystine Analog (CA) in this kit can be taken up into cells via xCT, and the incorporated CA can be specifically detected using the Fluorescent Probe and Reducing Agent. Thus, the xCT activity can be measured easily.[Patent applied]

The relevant technicals are published in the following journal:
Shimomura T, Hirakawa N, Ohuchi Y, Ishiyama M, Shiga M, Ueno Y, Simple Fluorescence Assay for Cystine Uptake via the xCT in Cells Using Selenocystine and a Fluorescent Probe. ACS Sensors, 2021, 6(6), 2125-2128

Application Data 1: Evaluation of xCT inhibitor Sulfasalazine or Erastin)

Using this kit, we measured the inhibitory effect of sulfasalazine and erastin on cystine uptake by HeLa cells.
The fluorescence intensity of the sulfasalazine and elastin groups decreased significantly, indicating that both reagents inhibit cystine uptake.

Experiment Condiitons
Cell Line: HeLa cells
Pretreatment: DMEM (cystine-free, serum-free), 37℃, 5 min
Uptake conditions：0.5 mmol/l sulfasalazine or 2 μmol/l erastin / Cystine Analog / DMEM (cystine-free, serum-free), 37℃, 30 min
Instrument: Fluorescent Plate Reader
Filter: Ex=485 nm, Em=535 nm

Application Data 2: Comparison with Radio Isotope Method

Cystine uptake assay, which used to require RI measurement, can now be easily measured with a plate reader. We compared the uptake in Wild-type (WT), xCT-knockout (KO), and xCT-overexpressed (OE) cells, using Cystine Uptake Assay Kit and Radio Isotope method using [14C]-labeled cystine. As a result, in both cases, the uptake was decreased in KO and increased in OE compared to WT. Therefore, it was confirmed that this product correlated well with the Radio Isotope method.

*This data was kindly provided by Professor Hideyo Sato and Assistant Professor Mami Sato, Laboratory of Biochemistry and Molecular Biology, Department of Medical Technology, Faculty of Medicine, Niigata University.

(Using WT's result as a benchmark value)

Experimental Conditions
Cell Line: HT1080 cells (wild-type or xCT-knockout or xCT- overexpressed)

○Cystine Uptake Assay Kit
Pretreatment: PBS (+) (cystine-free, 0.1% glucose, 0.01% Mg²⁺, 0.01% Ca²⁺), 37 ℃, 5 min
Uptake Conditions: Cystine Analog / PBS(+) (cystine-free, 0.1% glucose, 0.01% Mg²⁺, 0.01% Ca²⁺), 37℃, 30 min

○Radio Isotope Method
Pretreatment: None
Uptake Conditions: 0.05 mmol/l cystine + [¹⁴C]cystine (7.4 kBq/ml) /PBS(+) (0.1% glucose, 0.01% Mg²⁺, 0.01% Ca²⁺), 37℃, 2 min

Application Data 3: Evaluation of intracellular uptake and redox level after treating with Erastin

Erastin is a known inducer of ferroptosis. By inhibiting the cystine transporter (xCT), erastin inhibits the uptake of cystine. Cystine is the raw material for GSH. Therefore, Erastin ultimately decreases the amount of GSH. Decreased GSH then results in lipid peroxide accumulation and induction of ferroptosis.
The following experimental examples show changes in each aforementioned index as a consequence of erastin stimulation. Measurements are made using Dojindo reagents.

Using erastin-treated A549 cells, we measured intracellular Fe²⁺, ROS, lipid peroxide, glutathione, glutamate release into the extracellular space, and cystine uptake. As a result, inhibition of xCT by elastin was observed and also the release of glutamate and uptake of cystine were decreased. Furthermore, elastin treatment decreased intracellular glutathione while it increased intracellular Fe²⁺ , ROS, and lipid peroxides.

Cell Line: A549
Incubation Conditions: 100 μmol/l Erastin/MEM, 37℃, 3h