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

A natural killer cell and evaluation method technology, which is applied in the field of evaluation of the immune activity of natural killer cells in vitro, can solve the problems of unintuitive test results, increased unreliability of test results, and inability to directly repeatedly verify the accuracy of test results. Results are accurate and intuitive

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

AI Technical Summary

Problems solved by technology

[0006] However, these detection methods are all indirect methods, that is, the target cells are first labeled with a certain reagent, and then incubated with different concentrations of natural killer cells. When the target cells are attacked and damaged by natural killer cells, the permeability of the cell membrane Change, these marker substances are released into the supernatant, and the activity of natural killer cells is measured by measuring the release amount of the marker substances; through the indirect detection of an intermediate substance, the result is bound to be affected by the intermediate substance, resulting in an increase in the unreliability of the test results ; and the test results are not intuitive, and the accuracy of the test results cannot be directly and repeatedly verified. If the accuracy of the test results needs to be verified, it is necessary to use a microscope or other instruments to observe and corroborate

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

Examples

Experimental program
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Embodiment 1

[0084] This embodiment provides a method for evaluating the immune activity of natural killer cells in vitro, and the specific steps are as follows:

[0085] (1) Target cell markers:

[0086] Cultivate and collect target cells, add fluorescent dye CFSE (purchased from Biolegend, USA) to the target cells for incubation, label, and prepare the labeled target cells as a cell solution;

[0087] (2) Preparation of natural killer cells:

[0088] Collect fasting venous blood in the morning, put it in ethylenediaminetetraacetic acid (EDTA) anticoagulant sterile test tube, take 3mL blood sample and 3mL normal saline to mix and dilute, 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;

[0089] Centrifuge at 400g at room temperature for 30min. The centrifuge tube is divided into four layers. The top layer is plasma, the s...

Embodiment 2

[0121] This example provides a method for evaluating the immune activity of natural killer cells in vitro. The difference from Example 1 is that in this example, target cells and natural killer cells are cultured separately as a control group.

[0122] The specific steps are as follows:

[0123] (1) Target cell markers:

[0124] Cultivate and collect target cells, add fluorescent dye CFSE (purchased from Biolegend, USA) to the target cells for incubation, label, and prepare the labeled target cells as a cell solution;

[0125] (2) Prepare natural killer cells:

[0126] preparing natural killer cells as a cell solution;

[0127] (3) Carry out cell co-cultivation:

[0128] Add target cells and natural killer cells (as the experimental group) to the culture dish at the same time, and the effect-to-target ratio is set to 10:1. After incubation and co-cultivation, samples are taken, and Hoechst33342 (purchased from Thermofisher, USA) and PI dye (purchased from Sigma , USA), sta...

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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: carrying out mixed co-cultureon target cells carrying a fluorescence label A and natural killer cells, then adding a fluorescence label B to label the target cells and the natural killer cells, and adding a fluorescence label Cto carry out dyeing labeling on dead cells generated after co-culture; and then carrying out microscopic imaging on the cells by using different fluorescence channels, identifying and analyzing the cells in the same area in the microscopic image by combining an image synthesis analysis method, and evaluating the immunocompetence of the natural killer cells according to an analysis result. According to the evaluation method, multiple data such as the cell death rate, the cell self-injury rate and the cell specific killing rate can be directly obtained through the images, the immunocompetence ofthe natural killer cells is obtained according to the data, and interference of impurities and cell debris is effectively eliminated.

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] Natural killer cells (natural killer cells, NK cells) mainly exist in peripheral blood, spleen and bone marrow, and very little in lymph nodes. 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. Natural killer cells are closely related to the occurrence, development and treatment outcome of various diseases. Therefore, the evaluation of the immune activity of natural killer cells can provide a greater understanding of the cellular immune function of the body. [0003] The immune activity of natural killer cells can be used as one of the indicators to judge the bod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/10G01N21/64G01N33/50C12Q1/02
CPCG01N15/10G01N21/6458G01N33/5032G01N33/505G01N2015/1006
Inventor 罗浦文姜晶陈凯
Owner SHANGHAI RUIYU BIOTECH
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