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Fluorescence-based assay for detecting compounds for modulating the sodium-calcium exchanger (NCX) in 'forward mode'

A kind of compound, the technique of use, applied in the field of sodium-calcium exchange body and detecting its activity

Inactive Publication Date: 2010-01-27
SANOFI AVENTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The problem that arises in the prior art is therefore to identify an intensified assay with very good sensitivity and for high throughput screening and profiling of NCX modulators

Method used

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  • Fluorescence-based assay for detecting compounds for modulating the sodium-calcium exchanger (NCX) in 'forward mode'
  • Fluorescence-based assay for detecting compounds for modulating the sodium-calcium exchanger (NCX) in 'forward mode'
  • Fluorescence-based assay for detecting compounds for modulating the sodium-calcium exchanger (NCX) in 'forward mode'

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0074] 1. Measurement steps

[0075] 1.1 Assay reagents

[0076] The following chemical components were used as reagents for the assay:

[0077] Reagent

Chemicals

note

Assay buffer

3.5mM CaCl 2

133.8mM NaCl

4.7mM KCl

1.25mM MgCl 2

0.01%Pluronic F-127

10mM Hepes / NaOH pH 7.5

5mM Glucose

2.5mM probenecid

Add fresh at 1N on the day of use

1M probenecid in NaOH

(Probenecid) solution.

Dye Loading Buffer

Contains 2 μM Fluo-4 / AM

0.1%BSA

Assay buffer

Add 1 mM in DMSO

Fluo-4 / AM stock solution

compound buffer

Assay buffer

Multiple Compound Concentrations

Add 10 mM compound in DMSO

stock solution

ionophore solution

Contains 0.3% BSA

6 μM ionomycin

Assay buffer

Add 10 mM ion...

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Abstract

Carriers are an up-and-coming target family with enormous potential and offer commercial and scientific possibilities. The sodium / calcium exchanger is an important mechanism for the removal of Ca<2+> from various cells. In the heart, said sodium / calcium exchanger extrudes Ca<2+>, which has entered through Ca<2+> channels for the initiation of contractions, while Na<+> enters the heart cell. It is of considerable interest to identify compounds that modulate the activity of the sodium-calcium exchanger. The present invention relates to a fluorescence-based assay for the detection of compounds for modulating the NCX in 'forward mode'. The invention further relates to a parts set comprising cells that express NCX and to the use of said parts set for testing a compound for activity as an agonist or antagonist of NCX.

Description

technical field [0001] The invention relates to a sodium-calcium exchanger (sodium-calcium exchanger, NCX) and a method for detecting its activity. More specifically, the present invention relates to fluorescence-based assays for the detection of NCX modulating compounds in "forward mode". The invention also relates to a kit of parts comprising cells expressing NCX and uses of the kit. Background technique [0002] A fundamental need of life is compartmentalization - a principle with biofilms as a natural tool to achieve this. However, the lipid bilayer (the basic structure of cell membranes) is impermeable to most ions and compounds whose transport is essential to maintain vital functions of cells and organisms. The solution to this paradox relies on the semipermeable nature of the cell membrane—solutes that must cross the membrane are transported by specific membrane proteins. These transporters are responsible for generating and maintaining ion gradients, uptake of nut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N2500/10G01N33/6872
Inventor M·胡格T·利歇尔S·盖伯尔H·沃勒特
Owner SANOFI AVENTIS SA