Evaluation method of in-vitro natural killer cell immunocompetence and application thereof

A technology of natural killer cells and evaluation methods, which is applied in the field of in vitro natural killer cell immune activity evaluation, can solve the problems of large influence of subjective errors, unintuitive detection results, and inability to directly and repeatedly verify the accuracy of detection results, so as to achieve accurate detection results. and intuitive effects

Inactive Publication Date: 2021-02-02
SHANGHAI RUIYU BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain defects in flow cytometry. The detection process is greatly affected by the subjective errors of operators and analysts, and the detection results are not intuitive, so it is impossible to directly repeat the verification of the accuracy of the detection results. , it also needs to be corroborated by observation with a microscope or other instruments

Method used

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  • Evaluation method of in-vitro natural killer cell immunocompetence and application thereof
  • Evaluation method of in-vitro natural killer cell immunocompetence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] This example provides a method for evaluating the immune activity of natural killer cells in vitro.

[0073] Specific steps are as follows:

[0074] (1) Preparation of natural killer cells:

[0075] Mix and dilute 3mL blood sample with 3mL normal saline, take 4.5mL layered solution -1077 to a 15mL centrifuge tube, then carefully add 6mL of diluted anticoagulant blood to the layering solution along the tube wall, keeping the interface between the two clearly visible;

[0076] Centrifuge at 400g at room temperature for 30min. The centrifuge tube is divided into four layers. The top layer is plasma, the second layer is the required PBMC cells, the third layer is the separation solution, and the fourth layer is red blood cells. Use a 200μL pipette tip to carefully absorb the second layer. PBMC cells and transferred to 1.5mL EP tube, set aside;

[0077] (2) Mark tumor cells:

[0078] Tumor cells (K562 cell line) were collected and prepared into a cell concentration of 1...

Embodiment 2

[0114] This example provides a method for evaluating the immune activity of natural killer cells in vitro.

[0115] The difference from Example 1 is that in this example, target cells and natural killer cells were cultured separately as a control group.

[0116] The specific method is as follows:

[0117] After the prepared PBMC cells were mixed with the target cells, a control group was set at the same time, 100 μL medium was directly added to the target cells (as a target cell control group), and 100 μL medium was also added to the PBMC cells (as a natural killer cell control group). ), and cultured under the same conditions as the experimental group;

[0118] In the detection, the same detection and analysis methods were used as the experimental group.

[0119] In addition to the number of living target cells, the number of dead target cells, the death rate of target cells, the number of living natural killer cells, the number of dead natural killer cells, and the death r...

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Abstract

The invention provides an evaluation method of in-vitro natural killer cell immunocompetence and application thereof. The evaluation method comprises the following steps of: co-culturing a target cellcarrying a fluorescence label A and a natural killer cell, dyeing and labelling dead cells generated after co-culture by using a fluorescence label B, and carrying out microscopic imaging on the dyedand labelled cells by using a bright field, a fluorescence channel matched with the fluorescence label A and a fluorescence channel matched with the fluorescence label B respectively so as to obtaina microscopic image, recognizing cells in the obtained microscopic image through image recognition, performing superposition synthesis analysis on recognition results of the same region, and evaluating the immunocompetence of the natural killer cells according to an analysis result. According to the invention, a direct reading method for directly obtaining a detection result through an image is adopted, so that the detection result is more accurate and visual; and thus, the method is suitable for the fields of medical diagnosis and biomedical industrialization with higher standardization requirements.

Description

technical field [0001] The invention relates to the technical field of medical detection, and relates to a method for evaluating the immune activity of natural killer cells in vitro and an application thereof. Background technique [0002] The evaluation of the immune activity of natural killer cells (natural killer cells, NK cells) in vitro is important for the analysis of the etiology, treatment monitoring, prognosis and outcome of tumors, blood diseases, infectious diseases, immune diseases, organ transplantation, etc. guiding significance. NK cells mainly exist in peripheral blood, spleen and bone marrow, and the content of lymph nodes is very small. It is different from T cells and B cells. It does not need specific antibodies to kill target cells, nor does it require pre-sensitization of antigens. It can quickly kill and dissolve a variety of tumor cells or infected cells. [0003] NK cell activity can be used as one of the indicators to judge the body's anti-tumor a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/64G01N15/14
CPCG01N15/1434G01N21/6428G01N21/6458G01N21/6486G01N33/5032G01N33/505G01N2015/144G01N2021/6439
Inventor 罗浦文姜晶陈凯
Owner SHANGHAI RUIYU BIOTECH
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