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Flow Cytometry Assay for Natural Killer Cell Degranulation

A technology of natural killer cells and flow cytometry, applied in the biological field, can solve the problems that the test results are easily affected by the subjective factors of the tester, the detection speed is slow, and the accuracy is poor, so as to achieve small influence of human factors, high accuracy, Detect quick effects

Active Publication Date: 2018-03-02
倍科为(天津)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a flow cytometry detection method for natural killer cell degranulation, which can solve the problem of slow detection speed, low precision, poor accuracy, and detection results that are easily affected by the existing technology. The influence of subjective factors and other issues

Method used

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  • Flow Cytometry Assay for Natural Killer Cell Degranulation
  • Flow Cytometry Assay for Natural Killer Cell Degranulation
  • Flow Cytometry Assay for Natural Killer Cell Degranulation

Examples

Experimental program
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Embodiment 1

[0051] A flow cytometry detection method for natural killer cell degranulation, comprising the following steps:

[0052] S11. Separating the peripheral blood mononuclear cells in the sample to be tested and counting to adjust the cell concentration;

[0053] S12, taking the natural killer cell natural target cells and counting to adjust the cell concentration;

[0054] S13. Mix the peripheral blood mononuclear cells in step S11 and the natural killer cell natural target cells in step S12 in equal proportions by volume to obtain a cell mixture; incubate the cell mixture and centrifuge and discard Clear, get precipitation;

[0055] S14. Add flow staining buffer to resuspend the precipitate described in step S13, and add anti-CD3, CD56, CD107a flow antibodies and incubate at the same time;

[0056] S15. After the incubation is completed, centrifuge, and wash the precipitate with flow staining buffer;

[0057] S16. After washing, resuspend and mix the cells with flow cytometry ...

Embodiment 2

[0060] A flow cytometry detection method for natural killer cell degranulation, comprising the following steps:

[0061] S21. Separating the peripheral blood mononuclear cells in the sample to be tested and counting to adjust the cell concentration to 1.8×10 6 / ml;

[0062] S22. Take the natural killer cell natural target cell K562 and count to adjust the cell concentration to 1.8×10 6 / ml;

[0063] S23. Mix the peripheral blood mononuclear cells in step S21 and the natural killer cell natural target cells in step S22 in equal proportions by volume to obtain a cell mixture; store the cell mixture at 37°C, 5% CO 2 After co-incubating in the incubator for 2.5 hours, centrifuge at 1200 rpm for 6 minutes, discard the supernatant, and obtain a precipitate;

[0064] S24. Add flow staining buffer (PBS with 2.0% FBS and 2.0mM EDTA) to resuspend the precipitate described in step S23, and add flow cytometry antibodies against CD3, CD56, and CD107a and incubate at room temperature fo...

Embodiment 3

[0069] A flow cytometry detection method for natural killer cell degranulation, comprising the following steps:

[0070] S31. Separating the peripheral blood mononuclear cells in the sample to be tested and counting to adjust the cell concentration to 2.2×10 6 / ml;

[0071] S32. Take natural killer cell natural target cells P815 and count to adjust the cell concentration to 2.2×10 6 / ml;

[0072] S33. The peripheral blood mononuclear cells in step S31 and the natural killer cell natural target cells in step S32 are mixed in equal volume and divided into two groups on average, and anti-human CD16 antibody (antibody) is added to one of the groups. concentration of 0.25μg / 100μl) as the experimental group, and the other as the control group; 2 After co-incubating in the incubator for 3.5 hours, centrifuge at 1600 rpm for 4 minutes, discard the supernatant, and obtain a precipitate;

[0073] S34. Add flow staining buffer (PBS with 2.0% FBS and 2.0mM EDTA) to resuspend the preci...

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Abstract

The invention relates to the field of biotechnology, in particular to a flow cytometry detection method of natural killer cell degranulation. The method comprises the following steps of stimulating natural killer cell degranulation through a natural killing effect or an antibody-dependent cell-mediated cytotoxicity effect; then using flow cytometry to detect the rangeability of the proportion of vesicle membrane protein marker CD107a positive cells in natural killer cells in the total natural killer cells before and after stimulating degranulation so as to evaluate the degranulation capacity. The flow cytometry detection method of natural killer cell degranulation provided by the invention is quick in detection, large in flux, high in accuracy, and smaller in man-made influences. The stable and regulated technique process is established through optimization limitation carried out on key technology details such as natural stimulant selection, effect target cell matching and incubating time.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a flow cytometry detection method for natural killer cell degranulation. Background technique [0002] Natural killer cells (Natural Killer cells, NK cells) are important natural immune cells of the body. It plays an important role in the body's anti-tumor and anti-viral infection. And a large number of studies have shown that NK cells are involved in the pathological process of various inflammatory diseases and autoimmune diseases (such as liver inflammation and injury, habitual abortion). Therefore, the detection of NK cell function is essential for studying the pathogenesis of such diseases, assisting diagnosis and guiding treatment. [0003] The cytoplasm of NK cells contains high concentrations of cytotoxic granules in the form of vesicles. The ability of NK cells to degranulate is directly related to their cytotoxic activity. The detection of degranulation function reflects ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N15/14
CPCG01N15/14
Inventor 王昭
Owner 倍科为(天津)生物技术有限公司
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