PhosphoWorks™ Luminometric ATP Assay Kit *Extended Luminescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
PhosphoWorks™ Luminometric ATP Assay Kit *Maximized Luminescence* |
PhosphoWorks™ Luminometric ATP Assay Kit *DTT-Free* |
Overview | ![]() ![]() |
Platform
Luminescence microplate reader
Recommended plate | Solid white |
Components
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 ATP working solution (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 WORKING SOLUTION
1. Transfer the whole vial of 10 mL Reaction Buffer (Component C) into ATP Sensor (Component B) and mix well.
2. Add 20 µL of ATP Monitoring Enzyme (Component A) into the bottle of Component B+C and mix well to make ATP working solution. Note: Avoid potential ATP contamination from exogenous biological sources.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
Run ATP assay:
- 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°C, 5% CO2 incubator for a desired period of time, such as 24, 48 or 96 hours.
- Add 100 µL (96-well plate) or 25 µL (384-well plate) of ATP working solution into each well.
- Incubate at room temperature for 10 - 20 minutes.
- Monitor luminescence intensity with a standard luminometer.
Generate a standard ATP calibration curve:
An ATP standard curve should be generated together with the above assay if the absolute amount of ATP in samples needs to be calculated.
- Make a series of dilutions of ATP in PBS buffer with 0.1% BSA by including a sample without ATP (as a control) for measuring background luminescence. Note: Typically ATP concentrations from 1 nM to 10 µM are appropriate.
- Add the same amount of the diluted ATP solution into an empty plate (100 µL for a 96-well plate or 25 µL for a 384-well plate).
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of ATP working solution.
- Incubate the reaction mixture at room temperature for 10 to 20 minutes.
- Monitor the luminescence intensity with a standard luminometer.
- Generate the ATP standard curve.
Images

Citations
Authors: Chen, Yi and Liu, Chen and Zhou, Peng and Li, Jiannan and Zhao, Xiaoxiao and Wang, Ying and Chen, Runzhen and Song, Li and Zhao, Hanjun and Yan, Hongbing
Journal: Oxidative Medicine and Cellular Longevity (2021)
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Authors: Gonneaud, Alexis and Turgeon, Naomie and Boisvert, Frančois-Michel and Boudreau, Frančois and Asselin, Claude
Journal: FEBS letters (2015): 2776--2783
Authors: Qiu, Haifeng and Jackson, Am and a L , undefined and Kilgore, Joshua E and Zhong, Yan and Chan, Leo Li-Ying and Gehrig, Paola A and Zhou, Chunxiao and Bae-Jump, Victoria L
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Authors: Pohl, Kerstin and Hayes, Elaine and Keenan, Joanne and Henry, Michael and Meleady, Paula and Molloy, Kevin and Jundi, Bakr and Bergin, David A and McCarthy, Cormac and McElvaney, Oliver J and others, undefined
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Application notes
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