Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Inducer of neutrophil extracellular traps and rapid induction method thereof

A technology of neutrophils and inducers, which is applied in the fields of biological detection and medical detection to achieve the effect of good repeatability and shortening of induction time

Inactive Publication Date: 2019-01-01
张海英
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0021] In summary, although detecting the ability of cells to generate NETs under physiological or pathological conditions is very important for promoting or inhibiting the screening of NETs-related drugs or targeted therapeutic drugs, it is also of great significance in the field of biomedical testing, but there is currently a lack of rapid, Effective method for inducing NETs in vitro, especially human NETs

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Inducer of neutrophil extracellular traps and rapid induction method thereof
  • Inducer of neutrophil extracellular traps and rapid induction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] (1) Separation of human peripheral blood neutrophils:

[0075] Take 5ml of fresh EDTA or citrate anticoagulated peripheral venous blood from the subjects, and dilute it with PBS at a ratio of 1:1. Add 10 ml separation liquid Polymorphprep to 50ml centrifuge tube TM , and then slowly drip the diluted anticoagulated blood along the tube wall into the upper layer of the separation solution. Then, it was centrifuged horizontally at 700 × g for 30 min at room temperature. After centrifugation, there are different layers, from top to bottom are serum layer, nuclear cell layer, separation liquid layer, white blood cell (the vast majority of which are neutrophils) layer and red blood cell layer. Use the capillary to extend into the white blood cell layer, and gently suck out all the white blood cells along the tube wall to a new EP tube. Then wash with 1ml PBS, centrifuge at 1500rpm for 10min, collect the precipitate, and repeat once. Cells were resuspended in RPMI medium c...

Embodiment 2

[0087] (1) Separation of human peripheral blood neutrophils:

[0088] With embodiment 1 step (1).

[0089] (2) Induce neutrophils to form NETs:

[0090] (2.1) Dilute the extracted neutrophils with RPMI medium containing 5% FBS and adjust to a cell density of 1 × 10 6 a / ml;

[0091] (2.2) Divide the cells into 5 × 10 5 The number of cells / well was inoculated into a 24-well plate covered with glass slides, 0.5 ml per well;

[0092] (2.3) Using IgG4, APD and lipopolysaccharide to induce neutrophils in stages, the specific process is as follows:

[0093] S1: Pretreated cells: Add IgG4 to the wells to make the final concentration 20nM, incubate at 37°C, 5%CO 2 Place the pretreated cells in the incubator for 30 minutes;

[0094] S2: Rapid induction: APD was added to the above pretreated solution with a final concentration of 10 nM. At the same time, the temperature was raised from 37°C to 40°C, incubated for 15 minutes, and then cooled to 38°C at a rate of 0.5°C per minute. ...

Embodiment 3

[0099] (1) Separation of human peripheral blood neutrophils:

[0100] With embodiment 2 step (1).

[0101] (2) Induce neutrophils to form NETs:

[0102] (2.1) Dilute the extracted neutrophils with RPMI medium containing 5% FBS and adjust to a cell density of 1 x 10 6 a / ml;

[0103] (2.2) Divide the cells into 5 × 10 5 The number of cells / well was inoculated into a 24-well plate covered with glass slides, 0.5 ml per well;

[0104] (2.3) Use IgG4, APD and lipopolysaccharide composite inducer to directly induce neutrophils, the specific process is: add IgG4, APD and lipopolysaccharide directly to the well at one time, so that the final concentrations are 20nM, 10nM, 30nM at 37°C, 5%CO 2 Cells were induced for 90 min in an incubator.

[0105] (3) Post-processing: Quantitative analysis and identification of NETs:

[0106] The operation method is the same as step (3) of Example 2, and the extracellular fiber network structure can be seen in confocal laser microscopy imaging....

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides an inducer for in-vitro rapidly inducing neutrophil to generate extracellular traps and an induction method thereof. The inducer is prepared from the following components: a pretreatment component A which is selected from IgG2 and IgG4; an induction component B which is selected from APD or a mixture of APD and PMA; and lipopolysaccharide. Meanwhile, the invention also provides an inducation method for in-vitro rapidly inducing the neutrophil to generate extracellular traps (NETs). The induction method comprises the following steps: separating peripheral blood neutrophil; and using the inducer comprising the component A, component B and lipopolysaccharide to induce the neutrophil to form NETs. The inducer and induction method provided by the invention have good application prospect in the aspects for rapidly detecting mammal NETs and screening for promoting or inhibiting the NETs to generate a drug.

Description

technical field [0001] The present invention provides an inducer and an inducing method for inducing neutrophils to produce extracellular traps (NETs), specifically, a method for rapidly inducing neutrophils to produce extracellular traps in vitro for rapid detection. The compound inducer and the inducement method of the trapping net belong to the fields of biological detection and medical detection. Background technique [0002] Neutrophils are important effector cells in the innate immune system and the most abundant white blood cells in plasma. Neutrophils have a unique lobed nuclear structure, usually in the form of 3 to 5 lobes, so they are also called polymorphonuclear neutrophils (PMN). Under physiological conditions, neutrophils account for 50% to 70% of human peripheral blood leukocytes. It is the first cell recruited to the site of injury in response to pathogen invasion and is the first line of innate immune defense. When infection occurs, it is the first to be...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N5/0787
CPCC12N5/0642C12N2501/998C12N2501/999
Inventor 张海英
Owner 张海英
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com