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Cystine Uptake Assay Kit

Item # Unit Size
UP05-10
20 test
UP05-12
100 test

For Research Use Only Products

∼ Features ∼
– Easier way to cystine uptake assay
– Applied for plate assay

<Approximate usage> One plate of 96-well plate.

Contents:


Storage Condition: Store at -20oC; Non-medical deleterious substances
Shipping Condition: Ambient temperature

MSDS

The cystine transporter xCT is a crucial transporter that maintains cellular redox balance by regulating glutathione synthesis via cystine uptake. xCT is highly expressed in several types of cancer cells and is expected to be a therapeutic target for cancer.
Therefore, xCT has recently attracted researchers’ attention as one of the targets for cancer treatment. The xCT inhibitors sulfasalazine and erastin are known to reduce intracellular glutathione levels by inhibiting cystine uptake, thereby inducing ferroptosis, a form of cell death. It is also known that immune cells highly express xCT upon activation, suggesting that intracellular redox regulation via cystine uptake is also important for immune responses.

This kit uses Selenocystine as a Cystine Analog (CA) and can measure the cystine uptake ability of cells by a plate reader in a short time.


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
We evaluated the intracellular uptake and redox balance in A549 cells treated with erastin. As a result, we were able to get the three conclusions:
・To compensate for the loss of cysteine due to inhibition of cystine uptake, the uptake of amino acids increased.
・The Glucose uptake decreasing suggesting that metabolic reprogramming may have taken place.
・The decrease in glutathione due to inhibition of cystine uptake increased Fe2+, ROS, Lipid peroxides.




1 Amino Acid Uptake : Amino Acid Uptake Assay Kit (Code: UP04)
2 Glucose Uptake : Glucose Uptake Assay Kit-Green (Code: UP02)
3 Cystine Uptake : Cystine Uptake Assay Kit (Code: UP05)
4 Intracellular glutathione : GSSG/GSH Quantification Kit (Code: G257)
5 Intracellular labile Fe : FerroOrange (Code: F374)
6 Intracellular total ROS : ROS Assay Kit -Highly Sensitive DCFH-DA- (Code: R252)
7 Lipid Peroxides : Liperfluo (Code: L248)

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


Application Data 4: Evaluation by amino acid transporter inhibitor (T3)
Addition of T3, an LAT1 inhibitor, to HeLa cells inhibited BPA uptake into the cells in a concentration-dependent manner.



Which transporter uptake Cystine Analog into the cells?

Cystine Analog uptake via cystine/glutamate transporter (xCT).



How many cell lines have been tested by Cystine Uptake Assay Kit?

The following cell lines have been tested by Cystine Uptake Assay Kit:
A172, A549, A375, HCT116, HepG2, HL60, HT1080, MEF, SBC-5, U-251 MG.



Is the Cystine Analog degraded or metabolized after being uptake by cells?

Cystine Analog is very stable and will not be degraded during the experimental process.



Dose the cystine uptake solution can be stored for a period of time?

Cystine uptake solution cannot be stored. Prepare the proper amount of Cystine uptake solution before addition.



Can I use this kit to quantify the cystine taken by cells?

No, this kit can not quantify the cystine taken by cells.



What should I do if there is no change in the fluorescence signal?

Please consider extending the processing time of the cells with CA Uptake solution (30 minutes to 1 hour).



What should I do if the background fluorescence is high?

There might be some Cystine Analog remains in the well, Please use PBS wash once.



When preparing the CA Uptake solution may I use buffer in stead of cystine-free serum-free medium?

We have used HBSS or 0.1% Glucose PBS to prepare CA uptake solution when testing HeLa cells.



How can I correct the fluorescence intensity by the cell numbers?

You can use nucleic acid staining reagents to correct the Fluorescence intensity or use the Cell Count Normalization Kit (Code: C544) to count cell numbers and correct with them.



What kind of microplate can be used for this kit?

We have tested the following microplate:
Company Product Name Cat No.
ibidi μPlate 96 well ibiTreat black S 15 ib89626
AGC techno glass EZVIEW Glass Bottom Culture Plate LB 96well 5866-096
Thermo Fisher 96 Well Black/Clear Bottom Plate, TC Surface, Pack of 10 165305
No. Sample Instrument Reference(Link)
1) Cell
(A172; LN229)
Plate Reader K. Hayashima, H. Katoh, "Expression of gamma-glutamyltransferase 1 in glioblastoma cells confers resistance to cystine deprivation-induced ferroptosis", J. Biol. Chem., 2022, doi:10.1016/j.jbc.2022.101703.