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:10/12/2017)... ... October 12, 2017 , ... ... of a complex biological network, a depiction of a system of linkages and ... Dmitry Korkin, PhD, associate professor of computer science at Worcester Polytechnic Institute (WPI) ...
(Date:10/12/2017)... ... October 12, 2017 , ... ... with the addition of its newest module, US Hemostats & Sealants. , SmartTRAK’s ... hemostats, absorbable hemostats, fibrin sealants, synthetic sealants and biologic sealants used in surgical ...
(Date:10/11/2017)... ... October 11, 2017 , ... ... in its endogenous context, enabling overexpression experiments and avoiding the use of exogenous ... RNA guides is transformative for performing systematic gain-of-function studies. , This complement ...
(Date:10/11/2017)...  VMS BioMarketing, a leading provider of patient support solutions, ... Educator (CNE) network, which will launch this week. The VMS ... care professionals to enhance the patient care experience by delivering ... health care professionals to help women who have been diagnosed ... ...
Breaking Biology Technology:
(Date:4/18/2017)... SUNNYVALE, Calif. , April 18, 2017  Socionext Inc., a ... prototype of a media edge server, the M820, which features the ... face recognition software provided by Tera Probe, Inc., will be showcased ... and at the NAB show at the Las Vegas ... ...
(Date:4/11/2017)... Research and Markets has announced the addition of the ... ... grow at a CAGR of 30.37% during the period 2017-2021. ... prepared based on an in-depth market analysis with inputs from industry ... over the coming years. The report also includes a discussion of ...
(Date:4/5/2017)... , April 5, 2017 Today HYPR ... that the server component of the HYPR platform is ... providing the end-to-end security architecture that empowers biometric authentication ... HYPR has already secured over 15 million users across ... manufacturers of connected home product suites and physical access ...
Breaking Biology News(10 mins):