01 Cell Proliferation / Cell Cytotoxicity Assay

Amino Acid Uptake Assay Kit

Amino Acid Uptake Assay

Product code

UP04　 Amino Acid Uptake Assay Kit

Unit size	Price	Item Code
20 tests		UP04-10
100 tests		UP04-12

Component

20 tests	BPA Solution BPA Dilution Buffer Probe Solution	35 µl×1 35 µl×1 15 µl×1
100 tests	BPA Solution BPA Dilution Buffer Probe Solution	175 µl×1 175 µl×1 75 µl×1

C of A

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Description

Amino acids are essential for intracellular protein and nucleic acid synthesis, Especially for cancer cells which are proliferating continually. Since the supply of acetyl CoA from the glycolytic pathway is decreased in cancer cells, they have a furthermore huge demand for amino acids which are an important nutrient source for the TCA cycle. The research has shown that cancer cells increase the expression of the amino acid transporter LAT1 (L-type amino acid transporter 1) and take up a large number of amino acids, which is expected to be a new target for anti-Cancer drugs' discovery.
In immunotherapy, not only the metabolic changes in cancer cells but also in immune cells are considered to affect the treatment. For example, as senescence occurs in immune cells, their metabolic system changes, and their immune capacity to attack cancer cells decreases. Therefore, research is being conducted on nutrient uptake regulation by immune cells and improving the effectiveness of cancer immunotherapy.
After amino acid analogs (BPA) are taken up into cells via amino acid transporters, the fluorescent probe permeates the cell membrane and binds to the amino acid analogs, emitting fluorescence (λex=360 nm, λem=460 nm). This product is suitable for fluorescence imaging, plate reader, and flow cytometry, making it possible to visualize and quantify the amino acid uptake capacity of cells and is useful for evaluation of amino acids uptake capacity and screening of amino acids transporter inhibitors.

Manual

Manual

Technical info

Evaluate the inhibitor of amino acids transporter and compare the amino acids uptake capacity between different cell lines.

Simple Procedure

No need for washing steps.

Comparison with Existing Method

Compare to the existing method, the required time can be greatly reduced.

Method	Instrument	Time	Remarks
Amino Acid Uptake Assay Kit	Fluorescent Microscope Plate Reader Flow Cytometer	60 min	Simple procedure for amino acid analogs uptaking analysis
Radioisotope Labeling	Liquid Scintillation	2 Days	Allow testing different types of amino acids, but there are restrictions on use.
Metabolomics	LC/MS	3 hr	Allow testing different types of amino acids at the same time, but the result is hard to analyze.

Application Data: Evaluation of BCH (Inhibitor of amino acids transporter)

Evaluate the inhibition ability BCH (2-Aminobicyclo[2.2.1]heptane-2-carboxylic acid) in HeLa cells with Amino Acid Uptake Assay Kit.

＜Experimental Conditions＞
Cell Line: HeLa cells
Medium: MEM （5.5 mmol/l Glucose）
Incubation: 1 mmol/l BCH/HBSS (Hanks' Balanced Salt Solution), 37℃, 30 min
Instrument：Fluorescent Microscopy(Ex=340-380 nm, Em: 435-485 nm)
Instrument：Plate Reader (Ex=360 nm, Em: 460 nm)

＜Experimental Conditions＞
Instrument: Flow Cytometer (Ex=405 nm, Em: 425-475 nm)

References

Open References

No.	Sample Type	Instrument	Reference (Link)
1)	Cell SCC7 Cell	Flow Cytometer	T. Watanabe, Y. Sanada, Y. Hattori, and M. Suzuki, "Correlation between the expression of LAT1 in cancer cells and the potential efficacy of boron neutron capture therapy", 2022, J. Radiat. Res., doi:10.1093/jrr/rrac077.

Q & A

Q Which transporter take BPA into cells?: A

BPA has been reported to be uptaken via LAT1, LAT2 and ATB^0,+
(Reference: Wongthai P et al., “Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by ATB^0,+, LAT1 and LAT2”, Cancer Sci., 2015, Mar;106(3):279-86 )

In addition, Dojindo has conducted a competitive inhibition assay with BCH, an inhibitor of LAT1, and leucine, a substrate of LAT1. The results indicated that BPA uptake is inhibited.

Q Which cell lines has been analysis by this kit?: A

Adherent Cells include Hela, A549, HepG2, MCF-7, C2C12, MEF, U251.

Floating Cels include MOLT4.

Q Where is BPA localized in Cells?: A

BPA is uniformly present in the cells after permeating the cell membrane.

Q Is it possible to store the BPA solution and working solution for a period of time?: A

BPA solution and working solution cannot be stored.

Q What should I do if there is no difference in the fluorescence signal?: A

There are two possible causes, which may be improved by the following.

(1) The uptake capacity of the BPA uptake solution may be low. In this case, please consider increasing the concentration of the BPA Solution. (5 to 50 times dilution).

(2) The working solution may have expired. Please prepare the working solution again.

Q What should I do if the background is high?: A

There might be BPA remaining in the culture. In this case, please wash the cell once with HBSS.

Q Is it possible to quantitative evaluation with BPA?: A

It is not possible to quantitative the probe uptaken by cells. This product is used to evaluate the increase or decrease of amino acid uptake ability.

Q What kind of microplate can be used for fluorescence testing?

The following microplates have been used.

Company	Product Name	Cat No.
ibidi	μPlate 96 well ibiTreat black S 15	lb89626
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

Handling and storage condition

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Amino Acid Uptake Assay Kit

Amino Acid Uptake Assay Kit