ATP is a fundamental energy source and a lot of enzymes involved in the TCA cycle and oxidative phosphorylaion generate ATP. Therefore, determination of cellular ATP level is very crucial to understanding cellular metabolism. The ATP Determination Kit, provided by Invitrogen (cat# A22066), is a very useful assay kit to quantitative ATP by using recombinant firefly luciferase and its substrate D-luciferin in the cells. The assay is based on a simple luciferase assay which requires ATP as a co-factor for the enzymatic reaction to produce luminescent light (emission maximum is approximately 560 nm at pH 7.8). The luciferase assay is very sensitive (most luminometers can detect as little as 0.1 picomole of ATP). By using known amounts of an ATP standard, in vivo ATP levels can be quantified.
In addition to determining cellular ATP levels, the sensitivity of this lucifease has led to us to use it in numerous other applications, including detecting ATP production in various enzymatic reactions, such as ATPase and NADPH oxidase. One of the advantages of this kit is that it provides all of the reagents (20X concentrate of reaction buffer, dithiothreitol (DTT), firefly luciferase and D-luciferin) needed to perform this luminescence assay. We just simply need to mix the all of components with the sample and measure the ATP level. An ATP solution is also included for preparing standard curves for the quantification In addition, the D-luciferin (substrate) and purified firefly luciferase are packaged separately rather than premixed; the advantage of separated packaging is that researchers can optimize the proportions of D-luciferin and firefly luciferase for their particular system. This flexibility can be useful because the detection range of ATP will vary depending on instrument and sample requirements.
I have been used ATP determination kit to measure ATP level in human cell lines, such as HeLa and HEK293T cells, after knock-down or over-expression of my gene of interest. Usually, I run 5-10 different samples per experiment, with each samples run at least in triplicate. Therefore, my total number of samples per experiment is usually 15-30. Usually, I seed the cells on a 6-well plate and boil them in the plate to lyse the cells. For the best results, lysis of cell is critical. After boiling, the lysates are collected (via scraping), centrifuged at 14000 rpm for 5 min at 4°C, and then used in the ATP assay.