ReadiUse™ Rapid Luminometric ATP Assay Kit

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Adenosine triphosphate (ATP) plays a fundamental role in cellular energetics, metabolic regulation and cellular signaling. The quantitation of ATP can be used for a variety of biological applications. Because ATP is the energy source for almost all living organisms that rapidly degrades in the absence of viable organisms, its existence can be used to identify the presence of viable organisms. The measurement of ATP has been used for cell cytotoxicity, detection of bacteria on surfaces, quantification of bacteria in water, somatic cells in culture and food quality. The use of firefly bioluminescence to measure ATP was first proposed by McElroy when he discovered that ATP was essential for light production. Firefly luciferase is a monomeric 61 kD enzyme that catalyzes a two-step oxidation of luciferin, which yields light at 560 nm. The first step involves the activation of the protein by ATP to produce a reactive mixed anhydride intermediate. In the second step, the active intermediate reacts with oxygen to create a transient dioxetane, which quickly breaks down to the oxidized product oxyluciferin and carbon dioxide along with a burst of light. When ATP is the limiting component, the intensity of light is proportional to the concentration of ATP. Thus the measurement of the light intensity can be used for quantifying ATP using a luminometer. AAT Bioquest's ReadiUse™ Rapid Luminometric ATP Assay Kit (DTT free) comes with all the essential components in a ready-to-use format. It provides a fast, simple and homogeneous luminescence assay for monitoring cell proliferation and cytotoxicity in mammalian cells. This assay is based on the detection of ATP using firefly luciferase to catalyze the release of light by ATP and luciferin. It can be performed in a convenient 96-well or 384-well microtiter-plate format on a chemiluminescent microplate reader. The assay is extremely sensitive and can detect 50 cells/well. It has stable luminescent signal with half-life more than 2 hours. This ReadiUse™ Rapid Luminometric ATP Assay Kit does not use DTT, which eliminating the unpleasant odor.

Platform

Luminescence microplate reader

Recommended plate

Solid white

Example protocol

AT A GLANCE

Protocol Summary

Prepare cells (samples) with test compounds (100 µL/96-well plate or 25 µL/384-well plate)
Add equal volume of ready-to-use ReadiUse™ Rapid Luminometric ATP Assay Reagent (100 µL/96-well plate or 25 µL/384-well plate)
Incubate at room temperature for 10 - 20 minutes
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 STANDARD SOLUTION

ATP standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/21601

Optional, ATP is not provided.

Make 1 mM ATP stock solution with ddH2O or appropriate buffer, then perform serial dilution to achieve ATP concentrations ranging from 10 pM to 10 µM.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of ATP Standards and test samples in a white 96-well microplate. SD=ATP Standard, BL=Blank Control, TS=Test Sample

BL	BL	TS	TS
SD1	SD1	...	...
SD2	SD2	...	...
SD3	SD3
SD4	SD4
SD5	SD5
SD6	SD6
SD7	SD7

Table 2. Reagent composition for each well

Well	Volume	Reagent
SD1-SD7	100 µL	Serial Dilution of ATP (e.g. 10 pM to 10 µM)
BL	100 µL	ATP Assay Buffer (Component A)
TS	100 µL	Sample

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.

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

Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of ReadiUse™ Rapid Luminometric ATP Assay Reagent.
Incubate at room temperature for 10 - 20 minutes. Note: Aliquot and store the unused ReadiUse™ Rapid Luminometric ATP Assay Reagent at -20 ^oC, and avoid repeated freeze/thaw cycles.

Monitor the luminescence intensity with a standard luminometer.

Images

Figure 1. Cell number correlates with the luminescent signal. Different Jurkat cell number (2-fold dilution) was measured using the ReadiUse™ Rapid Luminometric ATP Assay Kit in a 96-well white plate using a ClarioStar plate reader (BMG Labtech). The kit can detect as low as 50 cells. There is a linear relationship (r2 >0.99) between the luminescent signal and cell number after 15 minutes or 2 hours incubation. The half-life of luminescent signal is more than 2 hours.

Figure 2. Cell number correlates with the luminescent signal. Different Jurkat cell number (2-fold dilution) was measured using the ReadiUse™ Rapid Luminometric ATP Assay Kit in a 96-well white plate using a ClarioStar plate reader (BMG Labtech). The kit can detect as low as 50 cells. There is a linear relationship (r2 >0.99) between the luminescent signal and cell number after 15 minutes or 2 hours incubation. The half-life of luminescent signal is more than 2 hours.

Citations

View all 13 citations: Citation Explorer

Homogeneous luminescent quantitation of cellular guanosine and adenosine triphosphates (GTP and ATP) using QT-LucGTP\&ATP assay
Authors: Kopra, Kari and Mahran, Randa and Yli-Hollo, Titta and Tabata, Sho and Vuorinen, Emmiliisa and Fujii, Yuki and Vuorinen, Iida and Ogawa-Iio, Aki and Hirayama, Akiyoshi and Soga, Tomoyoshi and others,
Journal: Analytical and Bioanalytical Chemistry (2023): 1--12

Guidelines for cell viability assays
Authors: Kamiloglu, Senem and Sari, Gulce and Ozdal, Tugba and Capanoglu, Esra
Journal: Food Frontiers (2020): 332--349

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, Fran&ccaron;ois-Michel and Boudreau, Fran&ccaron;ois 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