Extracellular ATP Assay Kit-LuminescenceE299 Product Manual

　Adenosine triphosphate (ATP), produced by glycolysis and mitochondria, is an important energy source for living cells. Meanwhile, ATP in the extracellular compartment is one of the damage-associated molecular patterns (DAMPs) released from damaged or dying cells, and extracellular ATP functions as an important messenger for cell-to-cell signaling events in neurotransmission, cell proliferation, inflammation, and immune response events. Recent studies have suggested that cellular senescence in human lung fibroblasts is induced through ATP released by oxidative stress. In addition, because extracellular ATP is released in the early stages of cell death, it is also attracting attention to elucidating the detailed mechanisms of cell death such as necroptosis and pyroptosis.
　The Extracellular ATP Assay Kit-Luminescence enables the detection of ATP in cell culture supernatant. This kit is formatted for 96-well microplates, allowing multiple sample measurements. 

Figure 1.　Principle of Extracellular ATP Assay Kit-Luminescence

Enzyme Solution	20 µl×1
Substrate	×1
eATP Assay Buffer	11 ml×1

Store at 0–5 °C

• Medium (serum-free)
• Microplate reader (luminescence)
• 96-well white microplate
• 20–200 µl multichannel pipette
• 100–1000 µl, 20–200 µl, 2–20 µl micropipettes
• Conical tube

• Equilibrate reagents to room temperature prior to use.
• Briefly centrifuge the tubes before opening them to ensure the content is at the bottom.
• Analysis of samples in triplicate is recommended for accuracy.
• The Substrate is stored in a glass bottle with an aluminum cap. Handle with caution, and wear gloves.
• Replacing the culture medium leads to release of ATP. Incubate cells for 3 hours after the medium is changed, then detect the ATP level (see Figure 2).
• Use a single micropipette when transferring cell culture supernatant to a 96-well white microplate.
• Refer to Figure 3 for precautions to take when measuring extracellular ATP.

Figure 2.　Effect of culture medium replacement
Figure 3.　Tips for using the extracellular ATP measurement kit

Preparation of a working solution

1. Add 1 ml of eATP Assay Buffer to the Substrate vial.
   Note: The Substrate vial is capped under vacuum pressure. Please open it carefully. Do not open quickly to avoid spilling contents.
2. Close the cap of the Substrate, and dissolve the contents completely, and transfer it to the eATP Assay Buffer bottle.
3. Add 20 µl of Enzyme Solution to the solution prepared in step 2).
   Note: Briefly centrifuge the Enzyme Solution tube before opening to ensure the content is at the bottom.
   Note: The working solution is stable for 1 month when stored at -20 °C.

Refer to Figure 4. Choose either experimental procedure ① or ② depending on the required experimental conditions.

Figure 4.　Selection guide for experimental procedure for extracellular ATP measurement

 

Experimental procedure ①－Experimental condition: drug treatment at one time point－

[For adherent cells]

Figure 5.　Experimental workflow for adherent cells

 

1. Seed cells in a microplate and culture overnight in an incubator (37 °C, 5%CO₂).
   Note: 2-5×10⁴ cells/well if using a 96-well microplate, 1.5-4×10⁵ cells/well if using a 24-well plate.
2. Remove the medium.
3. Add a serum-free medium and incubate for 3 hours in an incubator (37 °C, 5%CO₂).
   Note: Required amount of serum-free medium = 50 or 100 µl/well (96-well microplate), 250 µl/well (24-well plate).
   Note: If the medium contains serum, ATP released from cells is easily decomposed. We therefore recommend using serum-free medium. More information is posted on the product web page.
4. Add an equal amount of drug-containing serum-free medium as added in step 3), and culture in an incubator (37 °C, 5%CO₂).
5. Transfer the cell culture supernatants to a 96-well white microplate.
   Note: Refer to Table 1.
   Note: Please use a single micropipette.
   
   　　　　　　　　　Table 1.　Required amount of medium and transferred amount of cell culture supernatant

   	96-well microplate		24-well plate
   Amount of serum-free medium [Step 3)]	50 µl/well	100 µl/well	250 µl/well
   Amount of drug-containing serum-free medium [Step 4)]	50 µl/well	100 µl/well	250 µl/well
   Transferred amount of supernatant [Step 5)]	50 µl/well	50 or 100 µl/well	50 or 100 µl/well

6. Add 100 µl of working solution to each well.
   Note: Mix the contents using reverse pipetting to avoid air bubbles that could influence the luminescent signal.
   Note: After adding the ATP working solution, mix the contents for 2 minutes on an orbital shaker or similar.
   Note: Please protect the plate from light by covering it with aluminum foil because the luminescent signal is light sensitive.
7. Incubate the microplate at 25 °C for 10 minutes in a microplate reader.
   Note: Please incubate the microplate in an incubator which has been temperature-settled at 25 °C or room temperature around 25 °C.
   Note: The above incubation is necessary for a stable luminescent signal.
8. Measure luminescence with a microplate reader.

 

 

[For suspension cells]

Figure 6.　Experimental workflow for suspension cells

 

1. Prepare a cell suspension in a conical tube.
   Note: If the medium contains serum, ATP released from cells is easily decomposed. We therefore recommend using serum-free medium. More information is posted on the product web page.
2. Seed cells in a microplate and incubate for 3 hours in an incubator (37 °C, 5%CO₂).
   Note: 1-2×10⁵ cells/well if using a 96-well microplate (50 or 100 µl/well), 1-2×10⁶ cells/well if using a 24-well plate (250 µl/well).
3. Add an equal amount of drug-containing serum-free medium as seeded in step 2), and culture in an incubator (37 °C, 5%CO₂).
4. Transfer the cell culture supernatants to a 96-well white microplate.
   Note: Refer to Table 2.
   Note: Please use a single micropipette.
   
   　　　　　　　　　Table 2.　Required amount of medium and transferred amount of cell culture supernatant

   	96-well microplate		24-well plate
   Amount of cell suspension [Step 2)]	50 µl/well	100 µl/well	250 µl/well
   Amount of drug-containing serum-free medium [Step 3)]	50 µl/well	100 µl/well	250 µl/well
   Transferred amount of supernatant [Step 4)]	50 µl/well	50 or 100 µl/well	50 or 100 µl/well

5. Add 100 µl of working solution to each well.
   Note: Mix the contents using reverse pipetting to avoid air bubbles that could influence the luminescent signal.
   Note: After adding the ATP working solution, mix the contents for 2 minutes on an orbital shaker or similar.
   Note: Please protect the plate from light by covering it with aluminum foil because the luminescent signal is light sensitive.
6. Incubate the microplate at 25 °C for 10 minutes in a microplate reader.
   Note: Please incubate the microplate in an incubator which has been temperature-settled at 25 °C or room temperature around 25 °C.
   Note: The above incubation is necessary for a stable luminescent signal.
7. Measure luminescence with a microplate reader.

 

Experimental procedure ②－Experimental condition: drug treatment at multiple time points－

 

 

[For adherent cells]

Figure 7.　Experimental workflow for adherent cells

 

1. Seed cells in a microplate and culture overnight in an incubator (37 °C, 5%CO₂).
   Note: 2-5×10⁴ cells/well if using a 96-well microplate, 1.5-4×10⁵ cells/well if using a 24-well plate.
2. Remove the medium.
3. Add a serum-free medium and incubate for 3 hours in an incubator (37 °C, 5%CO₂).
   Note: Required amount of serum-free medium = 50 or 100 µl/well (96-well microplate), 250 µl/well (24-well plate).
   Note: If the medium contains serum, ATP released from cells is easily decomposed. We therefore recommend using serum-free medium. More information is posted on the product web page.
4. Add an equal amount of drug-containing serum-free medium as added in step 3) to “drug treatment for x hours” wells.
5. Add an equal amount of serum-free medium as added in step 3) to control wells.
6. Incubate for A hours in an incubator (37 °C, 5%CO₂).
7. Add an equal amount of drug-containing serum-free medium as added in step 3) to “drug treatment for y hours” wells.
8. Incubate for B hours in an incubator (37 °C, 5%CO₂).
9. Add an equal amount of drug-containing serum-free medium as added in step 3) to “drug treatment for z hours” wells.
10. Incubate for C hours in an incubator (37 °C, 5%CO₂).
11. Transfer the cell culture supernatants to a 96-well white microplate.
    Note: Refer to Table 3.
    Note: Please use a single micropipette.
    
    　　　　　　　　　Table 3.　Required amount of medium and transferred amount of cell culture supernatant

    	96-well microplate		24-well plate
    Amount of serum-free medium [Steps 3), 5)]	50 µl/well	100 µl/well	250 µl/well
    Amount of drug-containing serum-free medium [Steps 4), 7), 9)]	50 µl/well	100 µl/well	250 µl/well
    Transferred amount of supernatant [Step 11)]	50 µl/well	50 or 100 µl/well	50 or 100 µl/well

12. Add 100 µl of working solution to each well.
    Note: Mix the contents using reverse pipetting to avoid air bubbles that could influence the luminescent signal.
    Note: After adding the ATP working solution, mix the contents for 2 minutes on an orbital shaker or similar.
    Note: Please protect the plate from light by covering it with aluminum foil because the luminescent signal is light sensitive.
13. Incubate the microplate at 25 °C for 10 minutes in a microplate reader.
    Note: Please incubate the microplate in an incubator which has been temperature-settled at 25 °C or room temperature around 25 °C.
    Note: The above incubation is necessary for a stable luminescent signal.
14. Measure luminescence with a microplate reader.

 

 

[For suspension cells]

Figure 8.　Experimental workflow for suspension cells

 

1. Prepare a cell suspension in a conical tube.
   Note: If the medium contains serum, ATP released from cells is easily decomposed. We therefore recommend using serum-free medium. More information is posted on the product web page.
2. Seed cells in a microplate and incubate for 3 hours in an incubator (37 °C, 5%CO₂).
   Note: 1-2×10⁵ cells/well if using a 96-well microplate (50 or 100 µl/well), 1-2×10⁶ cells/well if using a 24-well plate (250 µl/well).
3. Add an equal amount of drug-containing serum-free medium as seeded in step 2) to “drug treatment for x hours” wells.
4. Add an equal amount of serum-free medium as seeded in step 2) to control wells.
5. Incubate for A hours in an incubator (37 °C, 5%CO₂).
6. Add an equal amount of drug-containing serum-free medium as seeded in step 2) to “drug treatment for y hours” wells.
7. Incubate for B hours in an incubator (37 °C, 5%CO₂).
8. Add an equal amount of drug-containing serum-free medium as seeded in step 2) to “drug treatment for z hours” wells.
9. Incubate for C hours in an incubator (37 °C, 5%CO₂).
10. Transfer the cell culture supernatants to a 96-well white microplate.
    Note: Refer to Table 4.
    Note: Please use a single micropipette.
    
    　　　　　　　　　Table 4.　Required amount of medium and transferred amount of cell culture supernatant

    	96-well microplate		24-well plate
    Amount of cell suspension [Step 2)]	50 µl/well	100 µl/well	250 µl/well
    Amount of drug-containing serum-free medium [Steps 3), 4), 6), 8)]	50 µl/well	100 µl/well	250 µl/well
    Transferred amount of supernatant [Step 10)]	50 µl/well	50 or 100 µl/well	50 or 100 µl/well

11. Add 100 µl of working solution to each well.
    Note: Mix the contents using reverse pipetting to avoid air bubbles that could influence the luminescent signal.
    Note: After adding the ATP working solution, mix the contents for 2 minutes on an orbital shaker or similar.
    Note: Please protect the plate from light by covering it with aluminum foil because the luminescent signal is light sensitive.
12. Incubate the microplate at 25 °C for 10 minutes in a microplate reader.
    Note: Please incubate the microplate in an incubator which has been temperature-settled at 25 °C or room temperature around 25 °C.
    Note: The above incubation is necessary for a stable luminescent signal.
13. Measure luminescence with a microplate reader.

Detection of extracellular ATP from Jurkat cells treated with staurosporine or doxorubicin

1. Jurkat cells were collected in a conical tube (1×10⁶ cells/ml in serum-free RPMI 1640 medium containing 1% penicillin-streptomycin) and cell suspension was prepared.
2. The Jurkat cell suspension prepared in step 1) was seeded in a 96-well microplate (1×10⁵ cells/well) and cultured for 3 hours in an incubator (37 °C, 5%CO₂).
3. RPMI (100 µl, serum-free) containing 1 µmol/l staurosporine or 5 µmol/l doxorubicin was added to 24-hour drug treatment wells.
4. RPMI (100 µl, serum-free) was added to control wells.
5. The cells were incubated for 17 hours in an incubator (37 °C, 5%CO₂).
6. RPMI (100 µl, serum-free) containing 1 µmol/l staurosporine or 5 µmol/l doxorubicin was added to 7-hour drug treatment wells.
7. The cells were incubated for 2 hours in an incubator (37 °C, 5%CO₂).
8. RPMI (100 µl, serum-free) containing 1 µmol/l staurosporine or 5 µmol/l doxorubicin was added to 5-hour drug treatment wells.
9. The cells were incubated for 2 hours in an incubator (37 °C, 5%CO₂).
10. RPMI (100 µl, serum-free) containing 1 µmol/l staurosporine or 5 µmol/l doxorubicin was added to 3-hour drug treatment wells.
11. The cells were incubated for 1.5 hours in an incubator (37 °C, 5%CO₂).
12. RPMI (100 µl, serum-free) containing 1 µmol/l staurosporine or 5 µmol/l doxorubicin was added to 1.5-hour drug treatment wells.
13. The cells were incubated for 1.5 hours in an incubator (37 °C, 5%CO₂).
14. Cell culture supernatant (50 µl) was transferred to a 96-well white microplate using a single micropipette.
15. The working solution (100 µl) was added to each well of the 96-well white microplate.
16. The microplate was orbitally shaken for 2 minutes in a microplate reader.
17. The microplate was incubated at 25 °C for 10 minutes in a microplate reader.
18. Luminescence was measured using a plate reader.

 

Figure 9.　Example of an assay plate format and workflow of drug treatment

 

Figure 10.　ATP release from Jurkat cells treated with staurosporine or doxorubicin

The ATP release behavior differed between staurosporine treatment and doxorubicin treatment.