Technical Data
A3999-20
ATP Assay Kit, BioAssay™
96Tests
Kits and Assays Storage: -20°CShipping: Dry Ice
Adenosine 5'-triphosphate (ATP) is the chemical energy for cellular metabolism and is often referred to as “energy currency" of the cell. ATP is produced only in living cells during photosynthesis and cellular respiration and consumed in cellular processes including biosynthetic reactions, motility and cell division. It is a key indicator of cellular activity and has been utilized as a measure of cell viability and cytotoxicity in research and drug discovery.

ATP Assay Kit provides a rapid method to measure intracellular ATP. The single working reagent lyses cells to release ATP, which, in the presence of luciferase, immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of intracellular ATP concentration.
Luciferase ATP+D-luciferin+O2 oxyluciferin+AMP+PPi+CO2+light

This non-radioactive, homogeneous cell-based assay is performed in microplates. The reagent is compatible with all liquid handling systems for high-throughput screening applications in 96-well and 384-well plates.

Key Features:
Safe. Non-radioactive assay.
Sensitive and accurate. As low as 0.02uM ATP or a single cell can be quantified.
Homogeneous and convenient. "Mix-incubate-measure" type assay.
No wash and reagent transfer steps are involved.
Robust and amenable to HTS: Z’ factors of > 0.5 are routinely observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems.

Applications:
ATP determination in cells and other biological samples.

Kit Contents:
Assay Buffer: 10ml
Substrate: 120ul
ATP Enzyme: 1 20ul
Standard: 100ul 3mM ATP
Storage conditions: store all reagents at -20°C. This kit is shipped on dry ice. Shelf life: 6 months after receipt.
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Assay Procedure:
Assays can be carried out in a tube or in a microplate.
1. Standard Curve. Prepare 500ul 30uM ATP Premix by mixing 5ul 3mM Standard and 495ul distilled water (for cell culture samples dilute ATP in culture media). Dilute standard as shown in the Table. Transfer 10ul standards into wells of a white opaque 96-well plate. Samples. Use 10ul sample per well in separate wells. For tissue samples, homogenize 20 mg sample in 200ul of cold phosphate-buffered saline, spin at 12,000 g for 5 min to pellet any debris. Transfer 1-10ul supernatant to each well and bring the volume to 10ul with PBS. Test several doses of the sample and choose the readings that are within the standard curve range for ATP Calculation:.
No Premix+H2O/media Vol (ul) ATP (uM)
1 50ul+0ul 50 30
2 40ul+10ul 50 24
3 30ul+20ul 50 18
4 20ul+30ul 50 12
5 15ul+35ul 50 9
6 10ul+40ul 50 6
7 5ul+45ul 50 3
8 0ul+50ul 50 0
For suspension cells, transfer 10ul of the cultured cells (103-104) into a white opaque 96 well plate. For adherent cells, culture 103-104 cells in white opaque microplate. At the time of assay, remove the culture medium immediately before adding 90ul Reconstituted Reagent (see below).
2. Assay. Bring Assay Buffer and Substrate to room temperature. Thaw enzyme on ice or at 4°C. Fresh Reconstitution is recommended. Store unused reagents including the enzyme at -20°C. For each 96-well, mix 95ul Assay Buffer with 1ul Substrate and 1ul ATP Enzyme. Add 90ul Reconstituted Reagent to each well. Mix by tapping the plate.
3. Read luminescence on a luminometer within 1 min after adding Reconstituted Reagent.

General Considerations:
Signal stability. Since the signal of the reaction decreases by ~1% each minute, for most accurate results, care should be taken that the time between adding the Reconstituted Reagent and luminescence reading is the same for all samples and standards.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
1. Schwarzer C., et al. (2008). Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells. Free Radic. Biol. Med. 45(12):1653-62.
2. Chandak P.G., et al. (2010). Efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase. J Biol. Chem. 285(26):20192-201.
3. Belleannée C., et al. (2010). Role of purinergic signaling pathways in V-ATPase recruitment to apical membrane of acidifying epididymal clear cells. Am. J. Physiol. Cell Physiol. 298(4): C817-C830.

Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.