PhosphoWorks™ Luminometric ATP Assay Kit *Steady Glow*

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
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

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.