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Multi-term separation method of neutrophil extracellular traps

A technology of neutrophils and separation method, which is applied in many separation fields of neutrophil external traps, and can solve the problems of neutrophil waste and inability to accurately separate neutrophil external traps, etc.

Pending Publication Date: 2022-01-18
YANAN HOSPITAL OF KUNMING CITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the inventors found in the research that this one-item conventional method cannot accurately separate the different components of neutrophil extracellular traps, which limits existing research, because the process of NETs generation does not always accumulate in the cells , and its products are divided into mitochondrial DNA complexes with better dissociation and nuclear DNA complexes with poor dissociation (spatial term), and the generation process of NETs has differences in induction time and formation components. As time goes on, the content of mitochondrial DNA complexes in the components will gradually increase (time item); the neutrophils that form NETs cannot be completely separated by conventional separation methods, resulting in waste of neutrophils. In addition, the products separated by conventional methods Not just nuclear DNA complexes and mitochondrial DNA complexes

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  • Multi-term separation method of neutrophil extracellular traps
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  • Multi-term separation method of neutrophil extracellular traps

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Use NETs separation method provided by the invention, the collection of the separated neutrophils NETs formed in 3h, the following process steps:

[0033] Sl: EP tube coating: 1ml volumes added into the EP tube, a concentration of 0.05mg / ml polylysine, 4 ℃ overnight incubation 8h above; After coating is completed in PH 7.4 phosphate buffered saline (PBS) washed twice, dried for use;

[0034] S2: immobilized cells: The isolated peripheral blood neutrophils was added 1ml EP After coating the tube, incubator at 37 ℃ IH; aspirated and adherent cells can not be replaced in 1ml of EP tube, of RPMI1640 + 10% fetal calf serum complete medium;

[0035] S3: an outer fishing net induced neutrophils: S2 is added to 1ml EP tube, 100ng / ml of phorbol myristate acetate (PMA) induced neutrophil outer fishing net generation;

[0036] S4: time trapping network entries neutrophils isolated: on the outer 3h after PMA induced neutrophil treated fishing net was separated complex;

[0037] S5:...

Embodiment 2

[0044] NETs using the separation method of the present invention provides for separation of collected NETs 5H neutrophils formed, steps are as follows:

[0045]S1: EP pipe package is added to the EP tube, a volume of 1 ml of a concentration of 0.05 mg / ml, incubation overnight at 4 ° C overnight; a pH is 7.4 phosphate buffer (PBS) Wash 2 times, dry and ready;

[0046] S2: Cell solid phaseization: 1 ml of neutral granulocytes isolated from peripheral blood added in an EP tube, 37 ° C under conditions, crankshavish culture tank culture tank; suction of cells that cannot be attached, replace the EP tube to 1 ml, rpmi1640 + 10% fetal bovine serum complete culture fluid;

[0047] S3: Neutraintenestylation: 1 ml, 100 ng / ml of Buddha (PMA) induced neutral granulocyteecell induced in the EP tube in S2;

[0048] S4: The neutral granulocyte trapping on time item is separated: a neutral granulocyte extravasation net of PMA-induced treatment is separated at 5 hours;

[0049] Separation of ...

Embodiment 3

[0055] The present embodiment is separated by a conventional method as a control of the separation method of the present invention, and the composite of the neutral granulocyte field is separated, the steps are as follows:

[0056] 1) 5% of CO into the neutralized blood culture liquid separated from peripheral blood 2 , 20 nm PMA incubated at 37 ° C for 4 h;

[0057] 2) After the incubation is completed, wash 2 times with a culture solution, with a serum-free culture solution, centrifuge at 300 F at 4 ° C for 10 min, and the upper level is to isolate the DNA complex of the resulting neutral granulocytes.

[0058] Fluorescent views of the composite of neutral granulocyte extravators are separated by conventional methods such as Figure 4 Indicated;

[0059] Result analysis:

[0060] According to Example 3 Figure 4 In Example 1 figure 2 And Example 2 image 3 The comparison can be obtained, and the composite of the neutral granulocellopatrophotion is separated from the two separation ...

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Abstract

The invention discloses a multi-term separation method of neutrophil extracellular traps. The multi-term separation method mainly comprises the following steps of 1) coating an EP tube, and immobilizing neutrophil in the EP tube; 2) inducing the neutrophil extracellular traps at different time points (time items); and 3) separating the induced neutrophil extracellular traps according to different distribution positions (spatial items) of required components. Through the separation method provided by the invention, the problem that a nuclear region DNA compound and a mitochondrial region DNA compound cannot be separated by existing neutrophil extracellular traps can be solved. In addition, the EP tube is coated with polylysine, so that the influence of cell debris in a centrifugation process can be avoided, and the purity of the neutrophil extracellular traps is improved. The purity of each component separated by the separation method provided by the invention can 95% or above.

Description

Technical field [0001] The present invention belongs to the field of biomedical research, more particularly it relates to a method for separating an outer neutrophils fishing net. Background technique [0002] Neutrophil outer netting (NETs) neutrophils stimulated by the activator is released to the extracellular one kind of network structure, myeloperoxidase (of MPO), citrullinated histones, neutrophil elastase protease protein to DNA mosaic skeleton. These proteins enriched in filaments formed by DNA, forming a spherical structure, these spherical aggregates of the protein concentration is generally higher than the concentration of the neutrophil proteins under physiological conditions, to exercise more efficiently their physiological functions - capture and kill destroy bacteria, fungi and viruses. After the procedure (NETosis) neutrophils fishing net forming the outer boot can capture not only pathogens, but also can enhance phagocytosis of leukocytes, it is possible to activ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0787
CPCC12N5/0642C12N2533/32C12N2501/999C12N2509/10
Inventor 孟平
Owner YANAN HOSPITAL OF KUNMING CITY