Description

Applications

The kit is intended for determination of intracellular ATP (adenosine triphosphate) in living cells. Extracellular ATP in the samples is enzymatically degraded. If the samples also contain somatic cells and the intention is to determine microbial ATP, we recommend 266-112 Microbial ATP Kit including a Cell Lysing Reagent for most animal cells. 266-311 ATP Biomass Kit can be used, if there is only one type of cells and no extracellular ATP in the samples. All living cells contain ATP where it plays the role of energy currency between different cellular processes. When cells die of natural causes, ATP is normally degraded. The intracellular concentration of ATP is carefully regulated to similar levels in all types of cells. ATP is therefore a good estimate of the total intracellular volume. Most bacterial cells contain approx. 2×10 ^-18 mol ATP per cell, while most eukaryotic cells, as a result of their larger size, contain 10 ^-15 mol ATP or more. ATP Biomass Kit HS, Intracellular ATP Kit HS and Microbial ATP Kit HS contain ATP Reagent HS (High Sensitivity) with a high luciferase activity. The luciferase activity in ATP Reagent HS is actually so high that if contaminated during reconstitution it will degrade the ATP background by one order of magnitude every 38 min. After mixing sample and ATP Reagent HS the light intensity decays by approx. 6% per min. This is slow enough to allow manual mixing and a tube luminometer without reagent dispenser can be used. The detection limit of the reagent is 10×10 ^-18 mol ATP with a sensitive luminometer1. This corresponds to 5 bacterial cells. We must here differentiate between bacterial cells and CFU, since one CFU may consist of several cells. If the detection limit is not adequate, it is possible to concentrate the sample by microfiltration, a procedure that will also remove most of the extracellular ATP.

Instruments

Any tube or microplate luminometer can be used. It should be noted that tube luminometers are about one order of magnitude more sensitive than microplate luminometers. If a microplate luminometer is used it should have minimum two dispensers and should be able to mix the microplate.

Assay procedure

Detailed described procedures are found in the “Instructions for Use”, which is included with every kit.

Product characteristics

The kit contains reagents for 150-375 assays (each vial of ATP Reagent HS is sufficient for 25 assays in a tube luminometer and 62.5 assays in a microplate luminometer).

Detection limit:

The detection limit of the reagent is 10×10-18 mol ATP with a sensitive luminometer ¹.

References

1 Lundin, A. (2000) Use of firefly luciferase in ATP related assays of biomass, enzymes and metabolites. Methods Enzymol. 305, 346-370.2 Lundin, A. (1999) ATP detection of biological contamination. In ”30th R3-Nordic Contamination Control Symposium” (Eds. G. Wirtanen, S. Salo and A. Mikkola), VTT, Espoo, Finland, pp. 337-352.