AAT Bioquest

PhosphoWorks™ Fluorimetric ATP Assay Kit

ATP dose response was measured with PhosphoWorks™ Fluorimetric ATP Assay Kit in a 96-well solid black plate using a Germini microplate reader (Molecular Devices).
ATP dose response was measured with PhosphoWorks™ Fluorimetric ATP Assay Kit in a 96-well solid black plate using a Germini microplate reader (Molecular Devices).
ATP dose response was measured with PhosphoWorks™ Fluorimetric ATP Assay Kit in a 96-well solid black plate using a Germini microplate reader (Molecular Devices).
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Adenosine triphosphate (ATP) plays a fundamental role in cellular energetics, metabolic regulation and cellular signaling. It is referred as the \"molecular unit of currency\" of intracellular energy transfer to drive many processes and chemical synthesis in living cells. ATP also serves as a signaling molecule for cell communication and plays an important role in DNA and RNA synthesis. AAT Bioquest offers a variety of bioluminescence assay kits to determine nanomolar (nM) range of ATP with recombinant firefly luciferase (Cat# 21610 & 21609). These kits require luminescence plate readers, are frequently used for cell viability or cytotoxicity assays. PhosphoWorks™ Fluorimetric ATP Assay Kit is based on a serial ATP-induced enzyme coupled reactions to produce hydrogen peroxide, which is spectrophotometrically quantified with our Amplite® Red Substrate. The assay can detect ~0.4 µM of ATP in a 100 µL reaction volume with minimal interference from ADP and AMP. It provides a robust, simple and convenient assay for measuring ATP levels in biological samples. The PhosphoWorks™ Fluorimetric ATP Assay is complementary to our luciferase-based ATP assay kits.


Fluorescence microplate reader

Excitation540 mn
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare ATP working solution (50 µL)
  2. Add ATP standards or test samples (50 µL)
  3. Incubate at room temperature for 10 - 30 minutes
  4. Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw the kit components at room temperature before starting the experiment.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. AmpliteTM Red Substrate stock solution (200X):
Add 30 µL of DMSO (Component E) into the vial of AmpliteTM Red Substrate (Component A) to make 200X AmpliteTM Red Substrate  stock solution.

2. ATP standard solution (10 mM):
Add 0.5 mL of ddH2O into the vial of ATP Standard (Component D) to make 10 mM ATP standard solution.


ATP standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/21620

Add 10 µL of 10 mM ATP standard solution into 990 µL 1X PBS buffer to generate 100 µM ATP standard solution (AS7). Take 100 µM ATP standard solution (AS7) and perform 1:3 serial dilutions to get serially diluted ATP standards (AS6-AS1) with 1X PBS buffer.


1. Add 5 mL of Assay Buffer (Component C) into Enzyme Mix bottle (Component B), and mix well.

2. Add 25 µL of 200X AmpliteTM Red Substrate stock solution to the Enzyme Mix bottle, mix well to make APT working solution.


Table 1. Layout of ATP standards and test samples in a solid black 96-well microplate. AS= ATP Standards (AS1 - AS7, 0.14 to 100 µM), BL=Blank Control, TS=Test Samples. 


Table 2. Reagent composition for each well. 

AS1 - AS750 µLSerial Dilutions (0.14 to 100 µM) 
BL50 µL1 X PBS Buffer 
TS50 µLtest sample
  1. Prepare ATP standards (AS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of ATP working solution to each well of ATP standard, blank control, and test samples to make the total ATP assay volume of 100 µL/well. For a 384-well plate, add 25 µL of ATP working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em=540/590 nm (Cutoff = 570 nm).



View all 48 citations: Citation Explorer
ATP-based cell viability assay is superior to trypan blue exclusion and XTT assay in measuring cytotoxicity of anticancer drugs Taxol and Imatinib, and proteasome inhibitor MG-132 on human hepatoma cell line HepG2
Authors: Nowak, E., Kammerer, S., Kupper, J. H.
Journal: Clin Hemorheol Microcirc (2018): 327-336
High-content image analysis (HCIA) assay has the highest correlation with direct counting cell suspension compared to the ATP, WST-8 and Alamar blue assays for measurement of cytotoxicity
Authors: Tahara, H., Matsuda, S., Yamamoto, Y., Yoshizawa, H., Fujita, M., Katsuoka, Y., Kasahara, T.
Journal: J Pharmacol Toxicol Methods (2017): 92-99
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)
NT1014, a novel biguanide, inhibits ovarian cancer growth in vitro and in vivo
Authors: Zhang, Lu and Han, Jianjun and Jackson, Am and a L , undefined and Clark, Leslie N and Kilgore, Joshua and Guo, Hui and Livingston, Nick and Batchelor, Kenneth and Yin, Yajie and Gilliam, Timothy P and others, undefined
Journal: Journal of Hematology & Oncology (2016): 91
The Different Effects of Atorvastatin and Pravastatin on Cell Death and PARP Activity in Pancreatic NIT-1 Cells
Authors: Chen, Ya-Hui and Chen, Yi-Chun and Liu, Chin-San and Hsieh, Ming-Chia
Journal: Journal of Diabetes Research (2016)
Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway
Authors: Yuan, Lingqin and Sheng, Xiugui and Willson, Adam K and Roque, Dario R and Stine, Jessica E and Guo, Hui and Jones, Hannah M and Zhou, Chunxiao and Bae-Jump, Victoria L
Journal: Endocrine-related cancer (2015): 577--591
BPA-induced DNA hypermethylation of the master mitochondrial gene PGC-1α contributes to cardiomyopathy in male rats
Authors: Jiang, Ying and Xia, Wei and Yang, Jie and Zhu, Yingshuang and Chang, Huailong and Liu, Juan and Huo, Wenqian and Xu, Bing and Chen, Xi and Li, Yuanyuan and others, undefined
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Loss of histone deacetylase Hdac1 disrupts metabolic processes in intestinal epithelial cells
Authors: Gonneaud, Alexis and Turgeon, Naomie and Boisvert, Frančois-Michel and Boudreau, Frančois and Asselin, Claude
Journal: FEBS letters (2015): 2776--2783
JQ1 suppresses tumor growth through downregulating LDHA in ovarian cancer
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
Journal: Oncotarget (2015): 6915
A neutrophil intrinsic impairment affecting Rab27a and degranulation in cystic fibrosis is corrected by CFTR potentiator therapy
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
Journal: Blood (2014): 999--1009


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