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Amino Acid Uptake Assay Kit

Item # Unit Size
UP04-10
20 tests
UP04-12
100 tests

For Research Use Only Products

Contents:


Storage Condition: Store at -20oC
Shipping Condition: Ambient temperature



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.



This product is developed with the technical guidance of Prof. Mitsunori Kirihata from Osaka Prefecture University.



Applications

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)


In addition, the fluorescence intensity of the dye was decreased in the presence of BCH (red) compared to the control (green) by flow cytometer, indicating that the amino acid uptake capacity was inhibited using BCH.



<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:Flow Cytometer (Ex=405 nm, Em: 425-475 nm)
Which transporter take BPA into cells?

We have confirmed that BPA uptake is inhibited by competitive inhibition experiments with BCH, an inhibitor of AT, and leucine, a substrate of LAT.


Which cell lines has been analysis by this kit?

Adherent Cells include Hela, A549, HepG2, MCF-7, C2C12, MEF, U251.
Floating Cels include MOLT4.


Where is BPA localized in Cells?

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


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

BPA solution and working solution cannot be stored.


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

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.


What should I do if the background is high?

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


Is it possible to quantitative evaluation with BPA?

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.


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