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PhosphoWorks™ Luminometric ATP Assay Kit *Bright Glow*

CHO-K1 cell number was measured with PhosphoWorks™ Luminescence ATP Assay Kit on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The integration time was 1 sec.
CHO-K1 cell number was measured with PhosphoWorks™ Luminescence ATP Assay Kit on a 96-well white plate using a NOVOstar plate reader (BMG Labtech). The integration time was 1 sec.
Impact of host-cell metabolism inhibition on ATP levels and τ2-NAD(P)H. Cellular ATP levels under glucose starvation and inhibition of oxidative phosphorylation by antimycin A. *ATP was measured using a luminometric ATP assay kit (ABD Bioquest, Sunnyvale, CA) and a microplate reader (Tecan Infinite 200 PRO, Maenedorf, Switzerland). HEp-2 cells were grown in 96-well plates (2000 cells/ well) and treated with the metabolic inhibitors as described above. ATP assays were performed according to the manufacturer's instructions. Source: Graph from <strong>Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell</strong> by Márta Szaszák, et al., <em>PLOS ONE</em>, July 2011. 
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Catalog Number21610
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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H-phraseH303, H313, H333
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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 can detect as low as 10 cells/well. 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


Component A: ATP Monitoring Enzyme1 vial
Component B: ATP Sensor (Light-sensitive)1 vial
Component C: Reaction Buffer1 vial (10 mL)

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 content of Reaction Buffer (Component C, 10 mL) 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 11 citations: Citation Explorer
Hepatitis B Surface Antigen Activates Unfolded Protein Response in Forming Ground Glass Hepatocytes of Chronic Hepatitis B
Authors: Li, Yao and Xia, Yuchen and Cheng, Xiaoming and Kleiner, David E and Hewitt, Stephen M and Sproch, Julia and Li, Tong and Zhuang, Hui and Liang, T Jake
Journal: Viruses (2019): 386
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