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PhosphoWorks™ Luminometric ATP Assay Kit *Extended Luminescence*

Adenosine triphosphate (ATP) plays a fundamental role in cellular energenics, metabolic regulation and cellular signaling. The PhosphoWorks™ ATP Assay Kit provides a fast, simple and homogeneous luminescence assay for the determination of cell proliferation and cytotoxicity in mammalian cells. The assay can be performed in a convenient 96-well and 384-well microtiter-plate format. The high sensitivity of this assay permits the detection of ATP in many biological systems, environmental samples and foods. This PhosphoWorks ATP Assay Kit has the stable luminescence signal as long as 4 hours. It has stable luminescence with no mixing or separations required, and formulated to have minimal hands-on time.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells (samples) with test compounds (100 µL/96-well plate or 25 µL/384-well plate)
  2. Add equal volume of ATP working solution (100 µL/96-well plate or 25 µL/384-well plate)
  3. Incubate at room temperature for 10 - 20 minutes
  4. 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:

  1. 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.

  2. Incubate the cell plate in a 37°C, 5% CO2 incubator for a desired period of time, such as 24, 48 or 96 hours.

  3. Add 100 µL (96-well plate) or 25 µL (384-well plate) of ATP working solution into each well.

  4. Incubate at room temperature for 10 - 20 minutes.

  5. 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.

  1. 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.

  2. 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).

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of ATP working solution.

  4. Incubate the reaction mixture at room temperature for 10 to 20 minutes.

  5. Monitor the luminescence intensity with a standard luminometer.

  6. Generate the ATP standard curve. 

Citations

View all 14 citations: Citation Explorer
Palmitic Acid Exerts Anti-Tumorigenic Activities by Modulating Cellular Stress and Lipid Droplet Formation in Endometrial Cancer
Authors: Zhao, Ziyi and Wang, Jiandong and Kong, Weimin and Newton, Meredith A and Burkett, Wesley C and Sun, Wenchuan and Buckingham, Lindsey and O’Donnell, Jillian and Suo, Hongyan and Deng, Boer and others,
Journal: Biomolecules (2024): 601
Knocking out Fkbp51 decreases CCl4-induced liver injury through enhancement of mitochondrial function and Parkin activity
Authors: Qiu, Bin and Zhong, Zhaohui and Dou, Longyu and Xu, Yuxue and Zou, Yi and Weldon, Korri and Wang, Jun and Zhang, Lingling and Liu, Ming and Williams, Kent E and others,
Journal: Cell \& Bioscience (2024): 1
Coronary Endothelium No-Reflow Injury Is Associated with ROS-Modified Mitochondrial Fission through the JNK-Drp1 Signaling Pathway
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)
KD025 Shifts Pulmonary Endothelial Cell Bioenergetics and Decreases Baseline Lung Permeability
Authors: Lee, Ji Young and Stevens, Reece P and Kash, Mary and Zhou, Chun and Koloteva, Anna and Renema, Phoibe and Paudel, Sunita S and Stevens, Troy
Journal: American Journal of Respiratory Cell and Molecular Biology (2020)
High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1
Authors: Wang, Mengqiao and Xu, Miao and Long, Yan and Fargue, Sonia and Southall, Noel and Hu, Xin and McKew, John C and Danpure, Christopher J and Zheng, Wei
Journal: Scientific Reports (2016)

References

View all 108 references: Citation Explorer
Cell-surface-localized ATP detection with immobilized firefly luciferase
Authors: Nakamura M, Mie M, Funabashi H, Yamamoto K, Ando J, Kobatake E.
Journal: Anal Biochem (2006): 61
Basic evaluation for new antimicrobial susceptibility testing of Mycobacterium leprae by bioluminescence assay (ATP method)
Authors: Yamazaki T, Gidoh M, Matsuoka M.
Journal: Nihon Hansenbyo Gakkai Zasshi (2006): 227
An efficient method for quantitative determination of cellular ATP synthetic activity
Authors: Hara KY, Mori H.
Journal: J Biomol Screen (2006): 310
Ca2+ oscillations stimulate an ATP increase during fertilization of mouse eggs
Authors: Campbell K, Swann K.
Journal: Dev Biol (2006): 225
Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase
Authors: Zamaraeva MV, Sabirov RZ, Maeno E, Ando-Akatsuka Y, Bessonova SV, Okada Y.
Journal: Cell Death Differ (2005): 1390
Page updated on December 13, 2024

Ordering information

Price
Unit size
10 Plates
1 Plate
Catalog Number
2160821609
Quantity
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Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Luminescence microplate reader

Recommended plateSolid white

Components

CHO-K1 cell number was measured with the PhosphoWorks™ Luminescence ATP Assay Kit on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The luminescence signal for CHO-K1 cells down to 100 cells per well was monitored for up to 2 hours (Z’ factor = 0.6). The integrated time was 1 second.
CHO-K1 cell number was measured with the PhosphoWorks™ Luminescence ATP Assay Kit on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The luminescence signal for CHO-K1 cells down to 100 cells per well was monitored for up to 2 hours (Z’ factor = 0.6). The integrated time was 1 second.
CHO-K1 cell number was measured with the PhosphoWorks™ Luminescence ATP Assay Kit on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The luminescence signal for CHO-K1 cells down to 100 cells per well was monitored for up to 2 hours (Z’ factor = 0.6). The integrated time was 1 second.
ATP dose response was measured with the PhosphoWorks Luminescence ATP Assay Kit. ATP concentrations from 10 uM to 0.1 nM was monitored for up to 5 hours at 1 second interval.