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PhosphoWorks™ Luminometric ATP Assay Kit *DTT-Free*

ATP dose response was measured with the PhosphoWorks™ Luminescence ATP Assay Kit *DTT-Free* on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The kit can detect (3 pmole/well) 0.03 nM ATP within 20 min. The integration time was 1 sec. The half life is more than 2 hours.
ATP dose response was measured with the PhosphoWorks™ Luminescence ATP Assay Kit *DTT-Free* on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The kit can detect (3 pmole/well) 0.03 nM ATP within 20 min. The integration time was 1 sec. The half life is more than 2 hours.
ATP dose response was measured with the PhosphoWorks™ Luminescence ATP Assay Kit *DTT-Free* on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The kit can detect (3 pmole/well) 0.03 nM ATP within 20 min. The integration time was 1 sec. The half life is more than 2 hours.
Jurkat cell number was measured with the PhosphoWorks™ Luminescence ATP Assay Kit *DTT-Free * on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The kit can detect as low as 100 cells. The integration time was 1 sec.
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


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 does not use DTT, and 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.

Platform


Luminescence microplate reader

Recommended plateSolid white

Components


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 kit components at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

1. Transfer 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 dilutions of ATP in PBS buffer with 0.1% BSA by including a sample without ATP (as a control) to measure background luminescence. Note: Typically ATP concentrations ranging from 0.1 nM to 1 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. Record the luminescence intensity with a standard luminometer.

  6. Generate the ATP standard curve.

Images


Citations


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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
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
Journal: Toxicology (2015): 21--31
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
G protein coupled receptor kinase 2 interacting protein 1 (GIT1) is a novel regulator of mitochondrial biogenesis in heart
Authors: Pang, Jinjiang and Xu, Xiangbin and Getman, Michael R and Shi, Xi and Belmonte, Stephen L and Michaloski, Heidi and Mohan, Amy and Blaxall, Burns C and Berk, Bradford C
Journal: Journal of molecular and cellular cardiology (2011): 769--776