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Culture method for regulatory T cells (Tregs) of mice by inducing of aggregation-induced emission luminogen (AIEgen) DPA-SCP

A technology of DPA-SCP and aggregation-induced luminescence, which is applied in the field of immunology research to achieve the effects of strong feasibility, high induction efficiency and low cost

Inactive Publication Date: 2019-11-08
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its regulation of immune cells has not yet been reported

Method used

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  • Culture method for regulatory T cells (Tregs) of mice by inducing of aggregation-induced emission luminogen (AIEgen) DPA-SCP
  • Culture method for regulatory T cells (Tregs) of mice by inducing of aggregation-induced emission luminogen (AIEgen) DPA-SCP
  • Culture method for regulatory T cells (Tregs) of mice by inducing of aggregation-induced emission luminogen (AIEgen) DPA-SCP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Anti-CD3 antibody coated plate

[0029] Dilute the 1 mg / mL anti-CD3 antibody stock solution 800 times with PBS to a final concentration of 1.25 μg / mL, take a 48-well plate and place it in 5% CO 2 , 37 ℃ incubator antibody incubation for 2h.

Embodiment 2

[0030] Example 2 mouse splenocyte extraction

[0031] 1. Kill the C57 / B6 mice by cervical dislocation, spray the whole body with alcohol, open the abdomen in the ultra-clean bench, take the spleen, and put it into 3-7mL 1640 medium containing 2% penicillin and streptomycin;

[0032] 2. Take a pair of frosted glass slides with one head and one side, take a small part of the spleen each time to the frosted place of the slides, grind them into single cells, suspend them in 3-7mL 1640 medium with 2% double antibody and culture them in the dish;

[0033] 3. Take a 200-mesh steel mesh and place it on a centrifuge tube, use a pipette to absorb the isolated splenocyte suspension, filter it through the steel mesh, and collect it in a centrifuge tube. Centrifuge at 1400r / min at 4°C for 7min, discard the supernatant;

[0034] 4. Add 1-2mL red blood cell lysate, resuspend the splenocyte pellet, place at room temperature for 5-7min, add 5-8mL1640 medium to the centrifuge tube to terminat...

Embodiment 3

[0036] Example 3 CD4 + CD25 + Regulatory T cell induction culture

[0037] 1. Divide the cultured cells into three groups, including anti-CD28 / TGF-β / IL-2 cytokine group (control group), anti-CD28 / TGF-β / IL-2 cytokine plus aggregation-induced luminescent molecule DPA -SCP group (AIE+Dark group), and anti-CD28 / TGF-β / IL-2 cytokine plus aggregation-induced luminescent molecule DPA-SCP plus light group (AIE+Light group); 0 day, the final concentration after its addition is shown in Table 1.

[0038] After 2.2 hours, take out the 48-well plate, absorb the anti-CD3 antibody liquid, and wash once with 1x PBS;

[0039] 3. Add the cells to the culture plate and place in 5% CO 2 , cultivated in a 37°C incubator for 24 hours;

[0040] 4. After 24 hours, except the control group, the other two groups were induced by adding the aggregation-induced luminescent molecule DPA-SCP, the volume ratio of which was 1:5000, and placed in 5% CO 2 , 37 ℃ incubator continued to cultivate for 24h. ...

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Abstract

The invention provides a culture method for regulatory T cells (Tregs) of mice by inducing of aggregation-induced emission luminogen (AIEgen) DPA-SCP. Multiple cell factors are used, AIEgen DPA-SCP isadded for illumination stimulation, and a large number of Tregs are induced from splenocytes of mice by induction amplification in vitro. The culture method is simple in induction process and high infeasibility; the culture method is short in cell culture period and low in cost; and the culture method is high in induction efficiency, good in repeatability and applicable to culture of Tregs. Compared with traditional methods, the culture method can more effectively induce a large number of CD4<+>CD25<+>Foxp3<+> Tregs, provides a convenient culture method for further study and has very good promotion values.

Description

technical field [0001] The invention belongs to the field of immunology research, and relates to a method for culturing mouse regulatory T cells induced by an aggregation-inducing luminescent molecule DPA-SCP. Background technique [0002] T cell (regulatory T cell, Treg) is CD4 + A subpopulation of T cells with important physiological roles in preventing the development of autoimmune diseases and maintaining self-tolerance. In 1995, Sakaguchi et al. obtained for the first time CD4 which has the function of maintaining autoimmune tolerance and immunosuppression + CD25 + Treg cells, this cell subset accounts for about 5% to 10% in peripheral blood of mice and humans. CD4 + CD25 + Treg cells mainly include innate Treg cells (nTreg) generated in the thymus, adaptive Treg cells (iTreg) induced by ordinary T cells, and endogenous induced Treg cells (pTreg) differentiated from antigen-stimulated T cells in peripheral tissues. , this type of cells plays an important role in m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N13/00
CPCC12N5/0637C12N13/00C12N2501/515C12N2501/51C12N2501/2302C12N2501/15C12N2501/999C12N2533/50
Inventor 常津丁丹高俊潇武晓丽杨涵陈星濛
Owner TIANJIN UNIV
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