The number of samples that can be recorded when the standard curve and sample are measured in triplicates. (50 tests/kit: 8 samples, 200 tests/kit: 48 samples; two times measurement for preparation of standard curves) Please refer to the instruction manual for a 96-well plate layout. 

A luminescence signal remains stable for 3 hours. However, luminescence is affected by temperature and light.  If you cannot measure luminescence immediately, please keep the plate in the dark and maintain a constant temperature of around 25℃.

Yes, you may. However, luminescence intensity decreases if you use black or transparent plates. Strong luminescent signals are detected in blank wells particularly in transparent plates. Therefore, it is strongly recommended to use white plates.

No.  You cannot store them because ATP is unstable.  Please add a Working solution to each well immediately after an experimental treatment and measure them within 3 hours. 

Yes. After the preparation, the Working solution is stable at - 20 ℃ for approximately 30 days. Avoid freeze-thaw cycles to prevent degradation of the solution; aliquot it into micro tubes and store at -20ºC. 

Yes. We have examples using mouse liver tissue.

General protocol is as below.

Extraction of ATP from liver samples via alkaline extraction method

1)Add cold 0.5 mol/l KOH solution to mouse liver (500 µl /100 mg).

    *Make sure blood is properly removed from the tissue sample, using hemoperfusion or other methods.

    *Any residual blood may affect the measurement.

2) Homogenize the tissue with Dounce tissue grinder.

3) Collect the sample and transfer it to a sample tube.

   Wash the tissue grinder with 500 µl of cold 0.5 mol/l KOH solution and transfer the solution into the sample tube. (Total volume: 1 ml)

4) Add 1 ml of cold ultrapure water to the sample tube, mix well, and put it on ice for 5 minutes. (Total volume: 2 ml)

    *If the solution is highly viscous, it may be difficult to separate after centrifugation in the next step.

     In this case, attach a needle (about 25 g) to a syringe and use the syringe to pipette the sample solution about 20-30 times until the   

     sample solution can be smoothly pipetted within the syringe.

5) Centrifuge at 12,000 x g , 4 °C for 5 minutes and collect each 900 µl of the supernatant in two new tubes.

Preparation of sample for ATP measurement

6) Add 200 µl of 1 mol/l KH₂PO₄ solution to 900 µl of the sample prepared in step 5. Mix well and put it on ice for 5 minutes.

7) Centrifuge at 12,000 x g for 5 minutes at 4°C and collect 1 ml of the supernatant in a tube as a sample for measurement.

*The extracted tissue samples cannot be stored. Please measure the sample within a day of the extraction.

*For dilution of the “standard” and “sample”, use a mixture of 0.5 mol/l KOH solution and 1 mol/l KH₂PO₄ solution at a ratio of 9:5 as the dilution solution.

ATP Level Changes in the Liver Tissue of NASH-Induced Mice

This kit uses firefly luciferin. Therefore, the luminescence measurement wavelength is 556 nm.