Method for detecting NK cytotoxic index based on flow cytometry

A technology of flow cytometry and NK cells, which is applied in the field of detection of NK cell killing activity based on flow cytometry, can solve the problems of inability to fully reflect the killing activity of NK cells, and achieve the effect of little interference or influence and a wide range of detection

Active Publication Date: 2019-09-13
XI AN JIAOTONG UNIV
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Problems solved by technology

Although they can specifically detect the apoptosis rate of target cells, they can only mark late apoptotic an

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  • Method for detecting NK cytotoxic index based on flow cytometry
  • Method for detecting NK cytotoxic index based on flow cytometry
  • Method for detecting NK cytotoxic index based on flow cytometry

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[0037] The present invention will be further described below in conjunction with the drawings:

[0038] See Figure 1 to Figure 4 , A method for detecting NK cell killing activity based on flow cytometry, including the following steps:

[0039] Step 1, inoculate and culture cells;

[0040] Step 2. Collect and stain the cells: use CD2-APC fluorescent dye to classify NK cells and tumor cells, and then use Annexin V-FITC / SYTOX Green to stain the apoptotic and necrotic target cells;

[0041] Step 3. Streaming computer detection.

[0042] Step 1 specifically includes: inoculate KHYG-1, K562 cells and culture them, exchange the cells 1-2 days before the experiment, adjust the cells to the optimal state of the logarithmic growth phase; KHYG-1 cells are cultured in cells containing IL-2 Medium.

[0043] Step 2 includes the following steps:

[0044] 1) Centrifuge NK cells and target cells at 1500rpm for 5 minutes, resuspend and count separately, adjust the cell density to 0.5х10 6 / ml;

[0045] 2...

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Abstract

A method for detecting NK cytotoxic index based on flow cytometry comprises the following steps of step 1, inoculating and culturing cells; step 2, collecting and dyeing the cells: classifying NK cells and tumor cells by using CD2-APC fluorescent dye, and then dyeing the apoptotic and necrotic target cells by using Annexin V-FITC/SYTOX Green; and step 3, carrying out flow type computer detection.The method provided by the invention uses Annexin V-FITC/SYTOX Green double dyeing and adopts the same detecting channel to mark all the apoptotic and necrotic cells, thereby obviously reducing the missed detection of various different states (early apoptosis, middle and late apoptosis or necrotic cells) of the tumor cells after NK cytotoxicity.

Description

technical field [0001] The invention belongs to the technical field of cell killing activity detection, in particular to a method for detecting NK cell killing activity based on flow cytometry. Background technique [0002] Natural killer cells (NK cells) are an important part of the body's natural immune system. Under the action of various cytokines (such as IL-2, IL-15, etc.) and feeder cells, NK cells can proliferate and activate, and then release perforin and granzymes to kill tumor cells, viruses and clear non-self cells. NK cells are closely related to tumor immunity, antiviral immunity, transplant immunity and autoimmune diseases. For example, in most tumors (especially those with metastases) and immunodeficiency patients, NK cell activity decreases; while in some viral infectious diseases, long-term use of interferon and its inducers, host resistance to transplantation, etc. In patients with drug reactions, NK cell activity increased. Therefore, accurate evaluati...

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Application Information

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IPC IPC(8): G01N15/14G01N21/64
CPCG01N15/1456G01N21/6428G01N2015/1081G01N2015/1486
Inventor 胡劲松张丹雷莉王爱英
Owner XI AN JIAOTONG UNIV
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