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Cell screening model of label-free Delta receptor

A label-free, cell-based technology, applied in the field of cell screening, can solve the problems of cumbersome operation, long experimental period, low throughput, etc., and achieve the effects of pathway integration research and simple operation, short experimental period, and high spatial and temporal resolution.

Pending Publication Date: 2020-06-30
TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have certain limitations, such as the traditional radioligand-receptor binding assay requires washing and filtration, long experimental cycle and low throughput, and this technique cannot distinguish receptor agonists and antagonists; the rest The detection method is mainly aimed at the activation of a certain signaling pathway, and often does not consider the activation of multiple pathways. It often requires fluorescent protein labeling or additional indicators, which makes the operation cumbersome, and the addition of these indicators will also have certain effects on the cells. damage, affecting the reliability of screening results

Method used

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  • Cell screening model of label-free Delta receptor
  • Cell screening model of label-free Delta receptor
  • Cell screening model of label-free Delta receptor

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: DMR characteristic signal spectrum of agonist enkephalin on HEK-293-Delta cells

[0032]Human embryonic kidney cells HEK-293-Delta cells were derived from a cell bank independently constructed by the laboratory, an inverted microscope was purchased from OLYMPUS, and enkephalin and naloxone were purchased from Tocris. The cell culture plate is an Epic optical biosensing 384 microwell plate, purchased from Corning, and the detection platform is the third generation of Corning Imager, the detected signal is the wavelength shift caused by cell dynamic mass reset (DMR).

[0033] HEK-293-Delta cells in the logarithmic growth phase were seeded in a cell-compatible 384 microwell plate, the medium used was DMEM (C11995503BT, GIBCO), the inoculation volume of each well was 40 μL, and the number of cells inoculated in each well was 2.0×10 4 Firstly, place the inoculated cell plate in a cell incubator and culture it for 20-22 hours until the cell confluency reaches ab...

Embodiment 2

[0034] Example 2: DMR characteristic signal spectrum of antagonist naloxone on HEK-293-Delta cells

[0035] HEK-293-Delta cells in the logarithmic growth phase were seeded in a cell-compatible 384 microwell plate, the medium used was DMEM (C11995503BT, GIBCO), the inoculation volume of each well was 40 μL, and the number of cells inoculated in each well was 2.0×10 4 Firstly, place the inoculated cell plate in a cell incubator and culture it for 20-22 hours until the cell confluency reaches about 95%, and conduct an activity test. Replace the cell culture solution in the microwell plate with Hank's balanced salt solution (containing 20mM HEPES), and add a volume of 30 μL to each well. After adding, place in Equilibrate on the imager for 1h; re-scan the baseline for 2min, add different concentrations of naloxone into the microwell plate, the volume of each well is 10μL, the concentration is 100000nM, 33333.33nM, 11111.11nM, 3703.7nM, 1234.57nM, 411.52nM . figure 2 . Studie...

Embodiment 3

[0036] Example 3: Desensitized DMR characteristic signal spectrum of HEK-293-Delta cells

[0037] HEK-293-Delta cells in the logarithmic growth phase were seeded in a cell-compatible 384 microwell plate, the medium used was DMEM (C11995503BT, GIBCO), the inoculation volume of each well was 40 μL, and the number of cells inoculated in each well was 2.0×10 4 Firstly, place the inoculated cell plate in a cell incubator and culture it for 20-22 hours until the cell confluency reaches about 95%, and conduct an activity test. Replace the cell culture solution in the microwell plate with Hank's balanced salt solution (containing 20mM HEPES), and add a volume of 30 μL to each well. After adding, place in Equilibrate on the imager for 1h; add different concentrations of enkephalins to the microwell plate to pretreat HEK-293-Delta cells for 1h, the volume of each well is 10μL, and the concentration is 10000nM, 3333.33nM, 1111.11nM, 370.37nM, 123.46nM , 41.15nM, 13.71nM, 4.57nM, 1.52n...

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Abstract

The invention provides a cell screening model of a label-free Delta receptor. On the basis of a label-free cell integration pharmacology technology, a method for screening an agonist and an antagonistof a Delta receptor is constructed by using a cell line stably expressing Delta. The method can be also applied to research on modifiers which affect downstream channels of the Delta receptor. The cell screening model has the characteristics of being free of damage, high in time-space resolution, high in sensitivity, high in flux, feasible in target-channel integration research, simple to operate, short in experiment cycle, and the like, labeling and addition of extra indicators are not needed in the detection process, functions of medicines at an overall level of living cells can be really answered, the discovery efficiency of the agonist, the antagonist and the channel modifiers of Delta can be greatly improved, great significances in describing pharmacological and physiological functions of Delta can be achieved, and meanwhile, instructions can be provided for screening on medicines with engagement of the Delta receptor for relieving pain and regulating related diseases such as gastrointestinal motility, moods, behaviors and heart and blood vessels.

Description

technical field [0001] The invention relates to the field of cell screening, in particular to a cell screening model of an unmarked Delta receptor. Background technique [0002] G protein-coupled receptor (GPCR) is the most important class of membrane receptors in cell signal transduction, and it is also one of the most concerned drug targets in the development of small molecule drugs, with about 34% of Modern drugs directly target this receptor family [Hauser, A.S., et al., Nature Reviews Drug Discovery 2017, 16, 829-842.]. The Delta receptor is an opioid receptor that belongs to the G protein-coupled receptor family. In 1977, Korsterlitz discovered an opioid receptor in the vas deferens of rats, named it Delta opioid receptor, and found that enkephalin is a relatively selective endogenous ligand of Delta opioid receptor; in 1992, Delta receptor was successfully cloned , which are distributed in the cortex, olfactory bulb, hippocampus, amygdala, basal ganglia and hypothal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12Q1/02
CPCC07K14/70567G01N33/502G01N33/5041C12N2510/00C12N2503/02G01N2500/10
Inventor 梁鑫淼薛珍珍王纪霞王志伟于广璞单彩龙
Owner TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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