Method of detecting specific killing effects of chicken spleen CD8<+>T cells based on flow cytometry

A flow cytometry and specific technology, which is applied in the field of detecting the specific killing effect of chicken spleen CD8+ T cells, can solve the problems of low sensitivity and strong radioactivity, and achieve short separation time, high cell activity and high sensitivity Effect

Active Publication Date: 2019-09-03
YANGZHOU UNIV
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Problems solved by technology

[0005] The object of the present invention is to target radioactive chromium ( 51 Cr) release assay cytotoxicity method has the disadvantages of low sensitivity and strong radioactivity, and est

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  • Method of detecting specific killing effects of chicken spleen CD8&lt;+&gt;T cells based on flow cytometry
  • Method of detecting specific killing effects of chicken spleen CD8&lt;+&gt;T cells based on flow cytometry
  • Method of detecting specific killing effects of chicken spleen CD8&lt;+&gt;T cells based on flow cytometry

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Embodiment Construction

[0029] The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

[0030] 1. Materials

[0031] 1.1 Experimental animals

[0032] Three-day-old SPF chickens were purchased from Jinan Spafas Poultry Company.

[0033] 1.2 Strains

[0034] Salmonella pullorum S06004 (Nal R ) was isolated and kept in our laboratory, collector: Geng Shizhong, contact information of the collector: 0514-87991556, collection location: Xuzhou, Jiangsu, collection time in March 2006; Salmonella pullorum mutant strain S06004Δ spiC The mutant strain was constructed and preserved in our laboratory (Liu Nannan. Construction of Salmonella pullorum S06004ΔspiC mutant strain and preliminary study on its immunobiological characteristics [D]. Yangzhou University, 2012.).

[0035] 2. Method

[0036] 2.1 Bacterial culture

[0037] Pick S06004, S06004Δ spiC A single colony was inoculated in 4 mL of LB liquid medium, and after 12 h ...

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Abstract

The invention relates to a method of detecting specific killing effects of chicken spleen CD8<+>T cells based on flow cytometry. The method comprises a first step of bacterial culture in which the bacteria are salmonella pullorum S06004 and S06004deltaspiC, a second step of chick immunization and sampling, a third step of target cell-macrophage preparation, a fourth step of effector cell-chicken spleen CD8<+>T cell preparation which comprises a substep of preparation of a chicken spleen lymphocyte suspension and a substep of magnetic bead sorting of a chicken spleen CD8<+>T cell and a fifth step of specifically killing macrophage by CTL. Compared with the prior art, the method disclosed in the invention has the following beneficial effects that the macrophage (target cell) obtained by adherence has high purity, the sorting effects for chicken spleen CD8<+>T cells are obvious, and the separation time is short; with the salmonella host cell- macrophages as the target cell, the salmonellaremoving ability by the host can be reflected more accurately; and the flow cytometry is used for chicken spleen CD8<+>T cell specific killing effects and can detect the CTL effects of the chicken spleen CD8<+>T cell in a single cell level.

Description

technical field [0001] The invention relates to a method for detecting chicken spleen CD8 based on flow cytometry + Methods of T cell-specific killing. Background technique [0002] Cellular immune response is mediated by T lymphocytes, which is conducive to promoting the production of antibodies in the body, and also helps to remove pathogenic microorganisms (such as bacteria, viruses, etc.) parasitic in the cells. In addition, cellular immune response also plays an important role in poultry immune surveillance and anti-tumor (such as Marek's disease) immunity. After poultry is infected by pathogenic microorganisms, T cells play a vital role in the immune response process by producing pro-inflammatory cytokines, regulating local and systemic immune responses, activating macrophages, and clearing intracellular bacteria. Among them, cytotoxic T cells (CTL) have lytic activity and play a key role in the recognition and removal of abnormal cells (such as tumors and pathogen-i...

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Application Information

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IPC IPC(8): G01N15/14C12N5/0783
CPCG01N15/14C12N5/0636G01N2015/0065C12N2509/00Y02A50/30
Inventor 焦新安唐娟潘志明胡茂志康喜龙孟闯顾丹王晶
Owner YANGZHOU UNIV
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