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Method of separating and purifying II-type innate lymphoid cells from animal lung tissue

A lymphocyte, separation and purification technology, applied in animal cells, cell dissociation methods, vertebrate cells, etc., can solve the problems of small number of ILC2 cells, lack of cell surface, and no description of the isolation method of mouse lung ILC2 cells.

Inactive Publication Date: 2017-05-17
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although major breakthroughs have been made in ILC2-related research, there are still major technical challenges in obtaining ILC2 cells for research due to the small number of ILC2 cells and the lack of highly specific marker molecules on the cell surface
Among them, the lung is one of the earliest organs where ILC2 cells were discovered. Lung ILC2 mediates the early type II immune response in diseases such as respiratory allergic reactions. Accurate isolation of ILC2 cells from mouse lungs is a key technology for studying ILC2 biology through mouse models , however, there is no exhaustive description of methods for isolating ILC2 cells from mouse lungs

Method used

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  • Method of separating and purifying II-type innate lymphoid cells from animal lung tissue
  • Method of separating and purifying II-type innate lymphoid cells from animal lung tissue
  • Method of separating and purifying II-type innate lymphoid cells from animal lung tissue

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Embodiment 1

[0033] The following experiments require aseptic technique.

[0034] (1) Normal or mouse lung tissue with lung inflammation was placed in sterile PBS and placed on ice.

[0035] (2) Cut the tissue into small pieces of about 1-2mm3, put the tissue piece into a centrifuge tube, add 5ml of collagenase I solution (0.1%, 1mg / ml, sigma) prepared by incomplete DM EM, tighten the cap and place Digest for 30 minutes at 220 rpm in a shaking box at 37°C (it can be seen that the tissue block has dispersed and lost the shape of the block, that is, the digestion is successful).

[0036] (3) After step 3 is completed, put it on ice to stop digestion, filter through a 200-mesh sieve, and gently grind the lung tissue remaining on the net before filtering.

[0037] (4) Centrifuge at 1060g at 4°C for 10 minutes, discard the supernatant. (The following centrifugation is at 4°C except that the step of density gradient centrifugation is at room temperature).

[0038] (5) Density gradient centrif...

Embodiment 2

[0052] The lungs of normal mice were digested with several different concentrations of collagenase I solutions prepared with physiological saline by the method of the present invention, and other operations were the same as in Example 1. The results showed that highly active type II innate lymphocytes could be isolated and purified. For the number of type II innate lymphocytes that can be obtained see Figure 4A .

[0053] After the lung inflammation was induced by 80 micrograms of blemisin intranasally in normal mice for one week, type II innate lymphocytes were isolated according to the method of Example 1. The results showed that highly active type II innate lymphocytes could be isolated and purified, and the resulting II Type innate lymphocyte count see Figure 4B .

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Abstract

The invention provides a method of separating and purifying II-type innate lymphoid cells from animal lung tissue. The method includes: taking the animal lung tissue, crushing, and adding I-type collagenase for digestion to obtain a lung tissue digestion solution; filtering the lung tissue digestion solution; separating to obtain lung tissue lymphoid cells through a Percoll method; using a II-type innate lymphoid cell specific antibody to mark the lung tissue lymphoid cells, and separating to obtain the II-type innate lymphoid cells through flow cytometry. By the method, high-activity II-type innate lymphoid cells can be separated and purified from mouse lung tissue.

Description

technical field [0001] The invention relates to a method for separating and purifying type II innate lymphocytes from animal lung tissue. Background technique [0002] In recent years, with the in-depth study of innate immune cells, a group of new cell subpopulations have been discovered. This subpopulation does not express antigen-specific recognition receptors (TCR or BCR), belongs to the lymphocyte lineage in development and morphology, and is called together with natural killer (NK) cells and lymphoid tissue-induced (LTi) cells discovered in the past. Innate lymphocytes (ILCs). According to the classification method of helper T (Th) cells, the ILC family can be divided into three categories according to the secretion of cytokines and the expression of transcription factors: ILC1, ILC2 and ILC3. [0003] Type II innate lymphoid cells (ILC2) is one of the above-mentioned newly discovered innate lymphoid cell populations, mainly distributed in the lungs, intestinal tract,...

Claims

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Application Information

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IPC IPC(8): C12N5/078
CPCC12N5/0651C12N2509/00C12N2509/10
Inventor 万晓春毕嘉成崔璐璐
Owner SHENZHEN INST OF ADVANCED TECH
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