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ATP Assay Kit-Luminescence

Item # Unit Size
A550-10
50 tests
A550-12
200 tests

For Research Use Only Products

∼ Features ∼
-High-sensitivity detection can be performed with stable luminescence.
-Measurement of unstable ATP is not necessary because ATP standard is supplied with the ATP Assay Kit-Luminescence.
-The procedure is very simple, only requiring the addition of the reagent.

Kit Contents:
50 tests(A550-10) 200 tests(A550-12)
Enzyme Solution 10 µl × 1 20 µl × 2
Substrate × 1 × 2
Assay Buffer 5.5 ml × 1 11 ml × 2
ATP Standard × 1 × 1

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


Adenosine triphosphate (ATP) is an energy source for living cells and is synthesized by both glycolysis and mitochondrial oxidative phosphorylation. Mitochondria generate 95% of cellular ATP, and mitochondrial dysfunction reduces ATP levels in cells. Therefore, the measurement of ATP levels has been established as a proxy for mitochondrial activity. Decreased ATP levels are associated with cancer, aging, neurodegenerative diseases, and mitochondrial diseases.
Cancer cells rely on glycolysis, a process that is less efficient than oxidative phosphorylation to synthesize ATP. However, recent studies have revealed that ATP synthesis in cancer cells shifts from glycolysis to oxidative phosphorylation when glycolysis is suppressed. The ATP Assay Kit-Luminescence enables the quantitation of intracellular ATP by luciferase luminescence assay. This kit can be applied for microplate assay.


Principle

The ATP Assay Kit-Luminescence enables the quantitation of intracellular ATP by luciferase luminescence assay. This kit can be applied for microplate assay.



Generate a stable luminescent signal with high reproducibility

Without complicated steps such as medium removal and cell washing, the ATP Assay Kit-Luminescence can generate a stable luminescent signal with a half-life of greater than 3 hours by simply mixing the kit components and adding the reagent. This kit contains an ATP Standard, which provides highly reproducible data without measuring unstable ATP.




Time-course measurements of relative light units. Data were collected from 0 to 3 hours after the addition of the reagent. HeLa cells (10,000cells/well)


Preparation of Standard Curve

ATP levels in a sample can be measured by a calibration curve established with ATP Standards included in this kit. If the ATP levels are greater than or equal to 2.5 µmol/l, the sample must be diluted before measurement.



Change in intracellular metabolism of rotenone-treated cells

Rotenone, which is known to inhibit the mitochondrial electron transport chain, was added to Jurkat cells, followed by measurement of intracellular ATP using the ATP Assay Kit-Luminescence. As a result, ATP production in the mitochondrial respiratory chain (the electron transport chain) was inhibited, and ATP concentrations were lower than those in the control cells.


Q. How many samples can I measure?
A. The number of samples that can be recorded when the standard curve and sample are measured in triplicates.
(50 tests/kit: 8 samples, 200 tests/kit: 48 samples; two times measurement for preparation of standard curves)
Please refer to the instruction manual for a 96-well plate layout.


Q. How about the stability of luminescence signals?
A. A luminescence signal remains stable for 3 hours. However, luminescence is affected by temperature and light. If you cannot measure luminescence immediately, please keep the plate in the dark and maintain a constant temperature of around 25℃.


Q. May I use color plates other than white plates for measurement?
A. Yes, you may. However, luminescence intensity decreases if you use black or transparent plates. Strong luminescent signals are detected in blank wells particularly in transparent plates. Therefore, it is strongly recommended to use white plates.


Q. Can I store my samples after an experimental treatment?
A. No. You cannot store them because ATP is unstable. Please add a Working solution to each well immediately after an experimental treatment and measure them within 3 hours.


Q. Can I store the Working solution?
A. Yes. After the preparation, the Working solution is stable at -20℃ for approximately 30 days. Avoid freeze-thaw cycles to prevent degradation of the solution; aliquot it into micro tubes and store at -20℃.