Navigation Links
ATP Quantitation in the LMax Microplate Luminometer (MaxLine Application Note #40)

Evelyn McGown, Ph.D. and Michael Su, M.S.
Molecular Devices Corporation, 8/00

Adenosine triphosphate (ATP) is present in all living cells. Because the level isstrictly-controlled, assay of ATP can be used as a indicator of viable cell number.ATP measurements are used to monitor raw materials and manufacturing planthygiene for bacterial contamination in the food, drug, health care and personalhealthcare industries, as well as for waste water analysis.1 In the biotechnologyand pharmaceutical industries, ATP measurements are used to evaluate cellproliferation and cytotoxicity. The bioluminescent assay of ATP has become verypopular because of its high sensitivity and convenience. In this application note,we show the ATP assay results obtained with the Molecular Devices Lmaxmicroplate luminometer.

The basis for the assay is the reaction catalyzed by luciferase, obtained from the common American firefly (Photinus pyralis).2 The enzyme catalyzes the ATP-dependent oxidation of luciferin with the concomitant release of light (Figure 1).When ATP is the limiting component in the reaction, the amount of light emittedis proportional to the concentration of ATP. The intensity of the emitted light canbe measured easily with the Lmax microplate luminometer.

1. Lmax microplate luminometer with SOFTmax PRO for Lmax (Molecular Devices Corp.)

2. ENLITENTM ATP Assay System, Cat. No. FF2000; Promega Corporation. 608277-2516. The kit contains Luciferase/Luciferin (L/L) reagent, ATP standard 10-7 M in HEPES buffer, pH 7.75) and ATP-free water.

3. Solid white 96-well microplates; e.g. Porvair PS white, E&G Wallac Cat. # 204003; Tel: 1-800-221-9650; or CorningCostar Cat. No. 3912; Tel: 1800492 1110). (In our hands, the Porvair PS plate has tended to have a lower background and slightly better sensitivity than the Costar plate.)

The instructions supplied with the kit (Technical Manual Part #TMF004)3 were followed, except for minor modifications to accommodate a 96-well format. NOTE: The technical manual also includes a section discussing sample preparation, including extraction of ATP from microorganisms or mammalian cells, which is not covered in this application note.

1. The M injector solvent delivery system was cleaned and sterilized by pumping 50% bleach through the injector and allowing it to stand for one hour, followed by rinsing with 50 mL deionized water. Then 75% ethanol was pumped through the system and allowed to stand for one hour, followed by a 100 mL deionized water rinse.

2. The enzyme and buffer solutions were thawed and kept on ice.

3. The L/L reagent was reconstituted by pouring the Reconstitution Buffer into the vial, gently swirling several times and allowing to stand at room temperature for one hour.

4. The solvent injection system was filled with L/L reagent by pumping 2.0 mL through it.

5. For fast kinetic assay to determine the best settings for endpoint assays, the Lmax was set to inject 100 uL (injector M) L/L reagent into selected samples of ATP dilutions (10 L/well) and then to immediately begin integrating at consecutive 0.1-sec intervals for a total of 10 seconds.

6. For determining sensitivity and dynamic range, the ATP standards were prepared by first diluting 100 L ATP stock standard with 900 L ATP-free water, followed by serial 1-to-10 dilutions in ATP-free water. The diluted standards (10 nM to 0.0001 nM) were kept on ice until use.

7. Diluted ATP standards (or ATP-free water blanks) were pipetted into micro-plate wells in triplicate (10 L/well). The final ATP content ranged from 0.01 to 1000 fmol/well.

8. For determining sensitivity and dynamic range, the Lmax was set up to inject 100 L/well, followed by a 2-second delay and a 1-second read.

9. The plate containing the ATP standards was placed into the drawer of the Lmax and the injection/read cycle initiated.

The reaction profile upon the addition of the luciferin/luciferase reagent is shown in Figure 2. The light emission in the presence of 100 fmol ATP/well (upper plot) was maximal within 1 seconds and remained at that level for at least 10 seconds. The blank (lower plot) showed essentially no response. Based on the data, we decided to use a delay of 2 seconds and then integrate total light for one second when reading an entire plate to determine sensitivity and dynamic range.

The standard curve from 0.01 to 1000 fmol/well is shown in Figure 3. The calculated limit of detection (amount producing a signal higher than 3 positive SD of the blank values above zero) was approximately 0.02 fmol/well. The dynamic range is at least 5 logs.

The Lmax microplate luminometer gives a detection limit of approximately 0.02 fmol ATP/well and a dynamic range of 5 logs with the ENLITEN ATP assay system. These results are as good as, if not better than, results obtained on a standard tube luminometer. The Lmax offers the advantage of a 96-well format and automated reagent addition for better precision and higher throughput. In addition, SOFTmax PRO for Lmax provides a powerful and convenient instrument control and data calculation package.


1. Jones, D. 1998. Bioluminescence assays using ATP. Luminescence Forum, 4: 1-9.

2. DeLuca, M.A. and W.D. McElroy, (1978) in Meth. Enzymol. vol 53:, p.3

3. Promega Corporation:

Note: Decontamination of the solvent delivery system is extremely critical for achieving satisfactory ATP assay results. Ethanol rinsing alone is not adequate. Not only must the system be sterilized, but any ATP remaining from killed bacteria must be destroyed in order to maintain blank values as low as possible.

back to top



Page: All 1 2 3 4

Related biology technology :

1. Quantitation of Cabergoline at Extremely Low Plasma Concentrations with a Triple Quadrupole Mass Spectrometer
2. Bioanalytical Method Intraday Validation for the Quantitation of Paroxetine in Bovine Plasma using the TSQ Quantum Mass Spectrometer
3. Quantitation of the Protein MIA as a Marker for Chondrocytes in Research Samples
4. Comparison of Different Methods of mRNA Quantitation
5. Quantitation of DNA for Automated Sequencing Using the VersaFluor Fluorometer
6. Quantitation of RNA for Hybridization Blots Using the VersaFluor Fluorometer
7. Quantitation of Lymphangiogenesis Using the iCycler iQ Real-Time PCR Detection System and Scorpions Detection System, Rev A
8. Quantitation of GAD67 Gene Expression in Prefrontal Cortex of Schizophrenia Patients Using the iCycler iQ Detection System and Molecular Beacons, Rev A
9. Benchmark Plus Microplate Reader: Quantitation of Protein Concentration Using Two Different Colorimetric Assay Kits, Rev A
10. A Robust LC/MS/MS Method for the Quantitation of Very Long Chain Fatty Acids for Research into the Characterization of Peroxisomal Disorders
11. A new LC/MS/MS Research Method for Rapid Quantitation of Five Immunosuppressant Drugs, including Mycophenolic Acid (MPA)
Post Your Comments:

(Date:6/24/2016)... (PRWEB) , ... June 24, 2016 , ... While the ... such as the Cary 5000 and the 6000i models are higher end machines that ... the height of the spectrophotometer’s light beam from the bottom of the cuvette holder. ...
(Date:6/23/2016)... June 23, 2016 /PRNewswire/ - FACIT has announced ... biotechnology company, Propellon Therapeutics Inc. ("Propellon" ... commercialization of a portfolio of first-in-class WDR5 inhibitors ... such as WDR5 represent an exciting class of ... precision medicine for cancer patients. Substantial advances have ...
(Date:6/23/2016)... , June, 23, 2016  The Biodesign Challenge (BDC), ... new ways to harness living systems and biotechnology, announced ... (MoMA) in New York City . ... participating students, showcased projects at MoMA,s Celeste Bartos Theater ... Antonelli , MoMA,s senior curator of architecture and design, ...
(Date:6/23/2016)... Apellis Pharmaceuticals, Inc. today announced positive ... its complement C3 inhibitor, APL-2. The trials were ... studies designed to assess the safety, tolerability, pharmacokinetics ... healthy adult volunteers. Forty subjects were ... dose (ranging from 45 to 1,440mg) or repeated ...
Breaking Biology Technology:
(Date:5/20/2016)... 20, 2016  VoiceIt is excited to announce ... By working together, VoiceIt and VoicePass ... and VoicePass take slightly different approaches to voice ... security and usability. ... new partnership. "This marketing and technology ...
(Date:5/9/2016)... -- Elevay is currently known as the ... high net worth professionals seeking travel for work   ... there is still no substitute for a face-to-face meeting. ... deal with a firm handshake. This is why wealthy ... citizenship via investment programs like those offered by the ...
(Date:4/28/2016)... 28, 2016 First quarter 2016:   ... compared with the first quarter of 2015 The gross ... M (loss: 18.8) and the operating margin was 40% (-13) ... Cash flow from operations was SEK 249.9 M (21.2) , ... is unchanged, SEK 7,000-8,500 M. The operating margin for ...
Breaking Biology News(10 mins):