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PhosphoWorks™ Luminometric ATP Assay Kit *Steady Glow*

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.
Ordering information
Price ()
Catalog Number21609
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
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 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


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 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 12 citations: Citation Explorer
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)
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

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
Ca2+ oscillations stimulate an ATP increase during fertilization of mouse eggs
Authors: Campbell K, Swann K.
Journal: Dev Biol (2006): 225
An efficient method for quantitative determination of cellular ATP synthetic activity
Authors: Hara KY, Mori H.
Journal: J Biomol Screen (2006): 310
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
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
Do rat cardiac myocytes release ATP on contraction
Authors: Godecke S, Stumpe T, Schiller H, Schnittler HJ, Schrader J.
Journal: Am J Physiol Cell Physiol (2005): C609
ATP bioluminescence assay for estimation of microbial populations of fresh-cut melon
Authors: Ukuku DO, Sapers GM, Fett WF.
Journal: J Food Prot (2005): 2427
Determination of ATP impurity in adenine dinucleotides
Authors: Pojoga LH, Haghiac ML, Moose JE, Hilderman RH.
Journal: Nucleosides Nucleotides Nucleic Acids (2004): 581
Identification of kinase inhibitors by an ATP depletion method
Authors: Singh P, Harden BJ, Lillywhite BJ, Broad PM.
Journal: Assay Drug Dev Technol (2004): 161
ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell
Authors: Satoh T, Kato J, Takiguchi N, Ohtake H, Kuroda A.
Journal: Biosci Biotechnol Biochem (2004): 1216