ATP Determination Kit - FAQs

The luminescence signal from the reaction has an emission peak of approximately 560 nm; however, luminometers and microplate readers in luminescence mode typically detect all wavelengths of light emission so you do not need to set any specific emission settings.

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The ATP Determination Kit is a 'flash' type of luciferase assay, so the luminescence signal will occur quickly and can decrease within a few minutes of starting the reaction. As a general guideline, you should incubate for at least 1-2 minutes from start of the reaction before reading the luminescence and the signal should be read in a time frame of under 5 minutes from reaction start. Using an automated microplate dispensing system or a multichannel pipette can allow you to add the ATP standards and samples to the reaction solution at the same time to control the timing of the reaction start.

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White, opaque, solid bottom microplates such as the Pierce 96-Well Polystyrene Plates, White Opaque (Cat. No. 15042) are recommended for the ATP Determination Kit and other luminescence assays.

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Cell lysis is required to release ATP from the cells to measure cellular ATP levels. Lysis buffer options that are compatible with the luciferase-luciferin reaction include the Pierce Luciferase Cell Lysis Buffer (2X) (Cat. No. 16189) and the 20X lysis buffer formula below. The 2X Pierce Luciferase Cell Lysis Buffer can be added to an equal volume of cells, while the 20X lysis buffer should be diluted 1:20 with water and used immediately for cell lysis prior to running the luciferase ATP assay. Cells are typically lysed at room temperature for 10-20 minutes before performing the ATP assay. The lysate is then used as a sample in the ATP Determination Kit (the cell lysate sample should be no more than 10% of the total assay volume). The cell lysis procedure and lysis buffer used may need to be optimized for the specific cell type since these buffers may not sufficiently lyse all cell types.

20X Lysis Buffer Recipe:
200 mM Tris (pH 7.5)
2 M NaCl
20 mM EDTA
0.2% Triton X-100

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Yes, the ATP Determination Kit (Cat. No. A22066) can be used for tissue samples like frozen heart tissue, as well as blood serum and plasma samples.

Please note, you must physically homogenize the tissue and then lyse the cells using a low-detergent lysis buffer.

We offer the following low-detergent lysis buffer:

Pierce Luciferase Cell Lysis Buffer (2X) (Cat. No. 16189)

As an alternative, you can make your own 20X buffer using this recipe:

20X Cell lysis buffer:
200 mM Tris, pH 7.5
2 M NaCl
20 mM EDTA
0.2 % Triton X-100

Reference:

Ahn BH, Kim HS, Song S, Lee IH, Liu J, Vassilopoulos A, Deng CX, Finkel T. A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14447-52. doi: 10.1073/pnas.0803790105. Epub 2008 Sep 15. PMID: 18794531; PMCID: PMC2567183.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.