Navigation Links
A highly sensitive ATP-luminescence assay using VICTOR2 with the dispenser option

INTRODUCTION

In high sensitivity flash type luminescence assays the measurement is done just after dispensing the activating reagent or after a short delay time. Over the past years luminescence substrates have been improved towards providing more stable signal. These so-called glow type substrates allow microplates to be counted without dispensors. However, flash type reagents will always have one advantage, sensitivity. In the glow type the luminescence signal is spread over a wide time scale (for example half life 30 min) and only a portion of it is read (1 - 10 s / well), so a lot of light is not detected. In flash assays most of the light is counted (half-life 5 s).

Adenosine triphosphate (ATP) is the energy reserve essential for viable cells. The concentration of ATP is stable in cells. Therefore the amount of ATP in the well correlates with the number of viable cells. ATP assays can be used for cell proliferation, cytotoxicity and cell adhesion studies. One application is the detection of microbial contamination in milk. In this study ATP samples were assayed with a Boehringer Mannheim flash type kit using VICTOR2 dispenser option. The detection is based on a light emitting (560 nm) reaction of ATP and luciferin in the presence of luciferase enzyme and oxygen.

METHODS

The assay of an ATP dilution series in a plate with a constant luciferase concentration was performed according to the kit instructions (ATP Bioluminescence Assay kit HS II, Boehringer Mannheim, Germany). 50 μL of Luciferase reagent was dispensed into white microplates (Combiplate, Labsystems, Finland) with 25 μL of ATP sample and 25 μL of cell lysis reagent. The wells were measured for 10 seconds after a 1 s delay.

RESULTS

The dilution series proved to be linear over 5 decades with a detection limit of 1.0E-11 M ATP, see figure 1. In the consistency measurement the intra-plate CV% was 3.2, with minimum and maximum differing only by 14% and 7% respectively from the average 21173 cps.

CONCLUSION

VICTOR2 with the dispenser option is an ideal instrument for microplate assays requiring immediate measurement after reagent injection. Applications are not restricted to luminescence assays but also cover fluorescence assays like Ca2+ ion channel studies.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Eppendorf Electroporation Application Notes Your participation is highly welcome !!!
2. Eppendorf Electroporation Application Notes Your participation is highly welcome !!!
3. Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 3000
4. Development of a sensitive enzyme fragment complementation assay for cyclic adenosine 3, 5 monophosphate and its validation for pharmacological screening at G protein-coupled receptors
5. The combination of monolithic columns and modern HPLC/MS/MS instrumentation for high chromatographic throughput and sensitive detection of beta-agonists
6. Enzyme Immunoassay for Studying Intracellular Levels of cAMP
7. LiquiChip Kits for bead-based cytokine and kinase assays
8. Quality control of antibodies using the 2100 bioanalyzer and the Protein 200 Plus assay
9. High-content screening of GPCR activation with MetaXpress, AcuityXpress and the Transfluor assay system
10. Bio-Plex Cytokine Immunoassays and ELISA: Comparison of Two Methodologies in Testing Samples From Asthmatic and Healthy Children, Rev A
11. Principles of Curve Fitting for Multiplex Sandwich Immunoassays, Rev B
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/24/2017)... Pa., Feb. 24, 2017  VWR Corporation (NASDAQ: VWR), the ... laboratory and production customers, today reported its financial results for ... Highlights: 4Q16 record quarterly ... 1.0% on an organic basis. ... an organic basis, while the Americas net sales increased 2.5%, ...
(Date:2/23/2017)... LONDON and NEW YORK ... at HIMSS, Lumeon , a leading digital ... (DN Telehealth), a provider of telemedicine and remote ... care pathways for telemedicine reimbursements.  ... and their patients, in real-time, extending consultations beyond ...
(Date:2/23/2017)... (PRWEB) , ... February 23, 2017 , ... ... Drug Discovery Services portfolio to include an array of biochemical analyses critical ... reliable data to drive their hit-to-lead and SAR programs, including inhibitor potency and ...
(Date:2/23/2017)... 2017  MIODx announced today that it has ... technologies from the University of California, San Francisco ... monitor a patient for response to immune checkpoint ... second license extends the technology with a method ... have an immune-related adverse event (IRAE) from their ...
Breaking Biology Technology:
(Date:2/8/2017)... Feb. 7, 2017 Report Highlights ... The global synthetic-biology market ... billion by 2021, growing at a compound annual growth rate ... overview of the global markets for synthetic biology. - Analyses ... 2016, and projections of compound annual growth rates (CAGRs) through ...
(Date:2/7/2017)... BEACH, New York , February 7, 2017 /PRNewswire/ ... known as ID Global Solutions Corporation [OTC: IDGS], ("Ipsidy" ... identity management and electronic transaction processing services, is pleased ... reorganization of the Company. Effective January 31, ... of the Board of Directors, CEO and President.  An ...
(Date:2/2/2017)... , Feb. 2, 2017  EyeLock LLC, a ... a new white paper " What You Should Know ... problem of ensuring user authenticity is a growing concern. ... authentication of users. However, traditional authentication schemes such as ... Biometric authentication offers an elegant solution to ...
Breaking Biology News(10 mins):