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PhosphoWorks™ Luminometric ATP Assay Kit *Extended Luminescence*

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.
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.
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H-phraseH303, H313, H333
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Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


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.


Luminescence microplate reader

Recommended plateSolid white


Example protocol


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.


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


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. 



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Ca2+ oscillations stimulate an ATP increase during fertilization of mouse eggs
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ATP bioluminescence assay for estimation of microbial populations of fresh-cut melon
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