Cell killing efficacy detection method and application thereof

A detection method and cell technology, applied in measuring devices, instruments, fluorescence/phosphorescence, etc., can solve problems such as inability to collect cell images, subjective errors of operators and analysts, difficulty in widespread use of flow cytometry, etc.

Active Publication Date: 2021-01-29
SHANGHAI RUIYU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of detecting cell killing, the method of flow cytometry needs to adjust the voltage according to experience in the experimental stage, and the gate needs to be based on experience in the data analysis stage. Such a process introduces subjective errors of operators and

Method used

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  • Cell killing efficacy detection method and application thereof
  • Cell killing efficacy detection method and application thereof
  • Cell killing efficacy detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080]This embodiment provides a method for detecting cell killing efficacy, and the specific steps are as follows:

[0081](1) Target cell labeling:

[0082]Cultivate and collect the target cells, add the fluorescent dye CFSE (purchased from Biolegend, USA) to the target cells, incubate, label, and prepare the labeled target cells into a cell solution;

[0083](2) Prepare effector cells:

[0084]Prepare effector cells into cell solution;

[0085](3) Co-cultivation of effective target cells:

[0086]Add target cells and effector cells to the culture dish at the same time, set the effect-to-target ratio to 1:1, take samples after incubation and co-cultivation, add Hoechst33342 (purchased from Thermofisher, USA) and PI dye (purchased from Sigma, USA), and stain;

[0087](4) Add the cells obtained in step (3) to the hemocytometer;

[0088](5) Place the loaded hemocytometer on the sample stage of the detection instrument, and capture the bright field channel, FL1 channel (matching fluorescent dye Hoechst33342)...

Embodiment 2

[0111]This embodiment provides a method for detecting cell killing efficacy, and the specific steps are as follows:

[0112](1) Target cell labeling:

[0113]Cultivate and collect the target cells, add the fluorescent dye CFSE (purchased from Biolegend, USA) to the target cells, incubate, label, and prepare the labeled target cells into a cell solution;

[0114](2) Prepare effector cells:

[0115]Prepare effector cells into cell solution;

[0116](3) Carry out cell co-culture:

[0117]Add target cells and effector cells (as the experimental group) to the culture dish at the same time, set the effect-to-target ratio to 1:1, take a sample after the incubation and co-cultivation, add Hoechst 33342 (purchased from Thermofisher, USA) and PI dye (purchased from Sigma, USA), dyeing;

[0118]Prepare control cells at the same time: add only target cells and culture medium to the culture dish as the target cell control group, and add only the effector cells and culture medium to the other culture dishes as the ef...

Embodiment 3

[0138]Compared with Example 2, in this example, different effective-to-target ratios (E:T) are also set as the experimental group to determine the killing efficacy of cells under different effective-to-target ratios.

[0139]Set the working concentration of effector cells to 1×106 / mL, 3×106 / mL, 6×106For different concentrations of / mL, the corresponding effect-to-target ratios are 1:1, 3:1 and 6:1.

[0140]The resulting overlay image is asFigure 5 As shown, different cells in the figure show different colors. When the effective target is relatively low, the number of live target cells is larger; when the effective target is relatively high, the dead target cells account for the majority. This result further illustrates that when the effective target is relatively high, the effector cells have better killing effect.

[0141]In summary, the method provided by the present invention is relatively intuitive, and can directly obtain visualized cell fluorescence images, obtain different states of c...

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Abstract

The invention provides a cell killing efficacy detection method and application thereof. The detection method comprises the following steps: carrying out mixed co-culture on target cells carrying a fluorescence label A and effector cells, then adding a fluorescence label B to label the target cells and the effector cells, and adding a fluorescence label C to carry out dyeing labeling on dead cellsgenerated after co-culture; and then respectively carrying out microscopic imaging on the cells by using different fluorescence channels, identifying and analyzing the cells in the same region in a microscopic image by combining an image synthesis analysis method, and obtaining the cell killing efficacy of effector cells according to an analysis result. Therefore, the detection method can effectively eliminate the interference of impurities and cell debris, can directly obtain a plurality of data such as cell death rate, cell self-injury rate and cell specific killing rate from the image, andhas the advantages of intuitive and visible detection result, high accuracy and high sensitivity.

Description

Technical field[0001]The invention relates to the technical field of cell killing activity detection, and relates to a method for detecting cell killing efficiency and its application.Background technique[0002]Killing function is an important aspect of the body's immune function. There are many kinds of effector cells with killing function in the immune system, such as natural killer cells (NK), cytotoxic T cells (CTL), monocytes with phagocytosis, and macrophages. Cells etc.[0003]At present, the detection methods of cell killing efficacy mainly include:51Cr release test, lactate dehydrogenase (LDH) release method and Calcein release test, etc. Among them, the classic method is51Cr release experiment, this method has good reproducibility, and is regarded as the "gold standard" of cell killing efficacy testing method. However, due to the use of isotopes to label target cells, there are many limiting factors such as short half-life, high requirements for isotope waste disposal and exp...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486Y02A50/30
Inventor 罗浦文姜晶陈凯
Owner SHANGHAI RUIYU BIOTECH
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