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:1/19/2017)... MD (PRWEB) , ... January 18, 2017 , ... ... the National Institutes of Health (NIH) to update its Data Sharing Policy. Specifically, ... “scoreable” element of grant applications subject to the existing policy. AMIA recommended that ...
(Date:1/19/2017)... 18, 2017  Northwest Biotherapeutics, Inc. (OTCQB: NWBO) ("NW ... therapies for operable and inoperable solid tumor cancers, announced ... Officer of NW Bio, will present at the Phacilitate ... the Hyatt Regency Hotel in Miami, Florida ... session entitled "New Therapeutic Approaches – Expanding the Reach ...
(Date:1/18/2017)... 2017   Boston Biomedical , an industry leader ... cancer stemness pathways, will feature data from two clinical ... 2017 ASCO Gastrointestinal Cancers Symposium, held from January 19-21, ... Napabucasin is an orally-administered investigational agent designed to ... Cancer stem cells (CSCs) possess the property of stemness ...
(Date:1/18/2017)... , ... January 18, 2017 , ... ... and more E&L expertise. Within Albany Molecular Research, Inc. (AMRI), the scientific staff ... past year and is planned for further growth in 2017. Extractable & Leachable ...
Breaking Biology Technology:
(Date:12/19/2016)... , España y TORONTO , 19 de diciembre ... Northern Biologics Inc. que permitirá el desarrollo acelerado de MSC-1, un ... en varios tipos de tumor en 2017, con múltiples sitios previstos ... ... clase con objetivo en el factor inhibidor de leucemia (LIF), una ...
(Date:12/15/2016)... and BADEN-BADEN, Germany , December ... leading global financial services provider, today announced an agreement with ... behavioural biometrics, to join forces. The partnership will enable clients ... strategies in compliance with local data protection regulation. ... In order to ...
(Date:12/15/2016)... , Dec. 15, 2016  There is much more ... or starting the engine. Continental will demonstrate the intelligence ... Las Vegas . Through the combination of the ... Entry) and biometric elements, the international technology company is ... personalization and authentication. "The integration of biometric ...
Breaking Biology News(10 mins):