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:2/10/2016)... (NYSE MKT: ISR), a medical technology company and innovator ... treatment of prostate, brain, lung, head and neck and ... second quarter and six months of fiscal 2016, which ... --> Revenue was $1.19 million for ... 31, 2015, a 12% increase compared to $1.07 million ...
(Date:2/10/2016)... ... February 10, 2016 , ... Benchmark Research, a fully-integrated ... of two long-standing principal investigators (PI) to the roles of Chief Medical Officer, ... , Dr. Laurence Chu, a Benchmark Research PI in the Austin office, will ...
(Date:2/10/2016)... ... February 10, 2016 , ... SonaCare Medical, ... and support program, Sonalinkā„¢ remote monitoring. The inaugural launch of this new technology ... 5th, connecting Dr. Samuel Peretsman to a HIFU technical expert at SonaCare Medical ...
(Date:2/10/2016)... (PRWEB) , ... February 10, 2016 , ... ... regenerative medicine, has announced a new agreement with Singapore-based Global Stem Cells Network ... from the Philippines, Thailand and Singapore in the latest adipose and bone marrow ...
Breaking Biology Technology:
(Date:2/1/2016)... -- Rising sales of consumer electronics coupled ... gesture control market size through ... electronics coupled with new technological advancements to drive global ... through 2020   --> Rising ... to drive global touchfree intuitive gesture control market ...
(Date:1/28/2016)... 28, 2016 Synaptics (NASDAQ: SYNA ), a leading ... second quarter ended December 31, 2015. --> ... fiscal 2016 increased 2 percent compared to the comparable quarter last ... fiscal 2016 was $35.0 million, or $0.93 per diluted share. ... for the first quarter of fiscal 2016 grew 9 percent over ...
(Date:1/25/2016)... 25, 2016  Glencoe Software, the world-leading supplier of ... industries, will provide the data management solution OMERO Plus ... Photo - ... Phenotypic analysis measures the characteristics and ... comparisons between states such as health and disease, the ...
Breaking Biology News(10 mins):