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ReadiUse™ Rapid Luminometric ATP Assay Kit
Adenosine triphosphate (ATP) plays a fundamental role in cellular energetics, metabolic regulation and cellular signaling. The quantitation of ATP can be used for a variety of biological applications. Because ATP is the energy source for almost all living organisms that rapidly degrades in the absence of viable organisms, its existence can be used to identify the presence of viable organisms. The measurement of ATP has been used for cell cytotoxicity, detection of bacteria on surfaces, quantification of bacteria in water, somatic cells in culture and food quality. The use of firefly bioluminescence to measure ATP was first proposed by McElroy when he discovered that ATP was essential for light production. Firefly luciferase is a monomeric 61 kD enzyme that catalyzes a two-step oxidation of luciferin, which yields light at 560 nm. The first step involves the activation of the protein by ATP to produce a reactive mixed anhydride intermediate. In the second step, the active intermediate reacts with oxygen to create a transient dioxetane, which quickly breaks down to the oxidized product oxyluciferin and carbon dioxide along with a burst of light. When ATP is the limiting component, the intensity of light is proportional to the concentration of ATP. Thus the measurement of the light intensity can be used for quantifying ATP using a luminometer. AAT Bioquest's ReadiUse™ Rapid Luminometric ATP Assay Kit (DTT free) comes with all the essential components in a ready-to-use format. It provides a fast, simple and homogeneous luminescence assay for monitoring cell proliferation and cytotoxicity in mammalian cells. This assay is based on the detection of ATP using firefly luciferase to catalyze the release of light by ATP and luciferin. It can be performed in a convenient 96-well or 384-well microtiter-plate format on a chemiluminescent microplate reader. The assay is extremely sensitive and can detect 50 cells/well. It has stable luminescent signal with half-life more than 2 hours. This ReadiUse™ Rapid Luminometric ATP Assay Kit does not use DTT, which eliminating the unpleasant odor.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare cells (samples) with test compounds (100 µL/96-well plate or 25 µL/384-well plate)
- Add equal volume of ready-to-use ReadiUse™ Rapid Luminometric ATP Assay Reagent (100 µL/96-well plate or 25 µL/384-well plate)
- Incubate at room temperature for 10 - 20 minutes
- Monitor the luminescence intensity
Important notes
To achieve the best results, it’s strongly recommended to use the white plates. Thaw all the kits components at room temperature before starting the experiment.
PREPARATION OF STANDARD SOLUTION
ATP standard
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/21601
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/21601
Optional, ATP is not provided.
Make 1 mM ATP stock solution with ddH2O or appropriate buffer, then perform serial dilution to achieve ATP concentrations ranging from 10 pM to 10 µM.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of ATP Standards and test samples in a white 96-well microplate. SD=ATP Standard, BL=Blank Control, TS=Test Sample
| BL | BL | TS | TS |
| SD1 | SD1 | ... | ... |
| SD2 | SD2 | ... | ... |
| SD3 | SD3 | ||
| SD4 | SD4 | ||
| SD5 | SD5 | ||
| SD6 | SD6 | ||
| SD7 | SD7 |
Table 2. Reagent composition for each well
| Well | Volume | Reagent |
| SD1-SD7 | 100 µL | Serial Dilution of ATP (e.g. 10 pM to 10 µM) |
| BL | 100 µL | ATP Assay Buffer (Component A) |
| TS | 100 µL | Sample |
- Treat cells (or samples) with test compounds by adding 10 µL of 10X compounds for a 96-well plate or 5 µL of 5X compounds for a 384-well plate in desired compound buffer. For blank wells (medium without the cells), add the corresponding amount of compound buffer.
- Incubate the cell plate in a 37 oC, 5% CO2 incubator for the desired period of time, such as 24, 48 or 96 hours.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of ReadiUse™ Rapid Luminometric ATP Assay Reagent.
- Incubate at room temperature for 10 - 20 minutes. Note: Aliquot and store the unused ReadiUse™ Rapid Luminometric ATP Assay Reagent at -20 oC, and avoid repeated freeze/thaw cycles.
- Monitor the luminescence intensity with a standard luminometer.
Citations
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Homogeneous luminescent quantitation of cellular guanosine and adenosine triphosphates (GTP and ATP) using QT-LucGTP\&ATP assay
Authors: Kopra, Kari and Mahran, Randa and Yli-Hollo, Titta and Tabata, Sho and Vuorinen, Emmiliisa and Fujii, Yuki and Vuorinen, Iida and Ogawa-Iio, Aki and Hirayama, Akiyoshi and Soga, Tomoyoshi and others,
Journal: Analytical and Bioanalytical Chemistry (2023): 1--12
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Journal: Analytical and Bioanalytical Chemistry (2023): 1--12
Guidelines for cell viability assays
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Journal: Food Frontiers (2020): 332--349
Page updated on November 21, 2024
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