Authors
Creative Bioarray
Summary
ATP is involved in a variety of enzymatic reactions to maintain normal life activities. When normal cells undergo apoptosis and necrosis, the content of ATP will be characteristic changed. Thus, ATP has been widely accepted as a valid marker of viable cells. The measurement of ATP using firefly luciferase is the most commonly applied method for estimating the number of viable cells.
The ATP cell viability assay utilizes luciferase to catalyze the formation of light from ATP and D-luciferin, which can be measured with a luminometer or Beta Counter. The advantage of ATP assay is that you do not have to rely on an incubation step with a population of viable cells to convert a substrate (such as tetrazolium and resazurin) into a colored compound. There is also no need to remove cell culture medium or wash cells before adding the reagent, which can be fully automatic for high throughput.
Introduction
For suspension cells, transfer 10 µL of the cultured cells (containing 103-104 cells) into luminometer plate. Add 100 µL of the Nuclear Releasing Reagent.
For adherent cells, remove culture medium and treat cells (103-104) with 100 µL of Nuclear Releasing Reagent for 5 minutes at room temperature with gentle shaking.
Add 1µL ATP Detection Cocktail into the cell lysate. Read the sample in 1 minute in a luminometer.
The background luminescence should be subtracted from all readings. The amount of ATP in experimental samples can then be calculated from the standard curve.
Note: The assay can be analyzed using cuvette-based luminometers or Beta Counters. The entire assay can also be done directly in a 96-well plate. It can also be programmed automatically using instrumentation with injectors.
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