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Apoptosis Detection Method and Apoptosis Detection Kit

A detection method and cell technology, applied in the field of cell detection, can solve the problems of inability to distinguish late cell apoptosis, low sensitivity, and inapplicability of high-throughput screening, etc.

Active Publication Date: 2021-12-14
GUANGZHOU RIBOBIO +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method cannot distinguish the state of late apoptosis and necrosis (necrosis), the sensitivity is not high, and it is only suitable for flow cytometry, not suitable for large-scale high-throughput screening
However, the TUNEL and DNA ladder methods can only analyze the cell subsets in late apoptosis, and the mitochondrial membrane potential analysis method cannot distinguish between early apoptosis and late apoptosis cells.

Method used

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  • Apoptosis Detection Method and Apoptosis Detection Kit
  • Apoptosis Detection Method and Apoptosis Detection Kit
  • Apoptosis Detection Method and Apoptosis Detection Kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Comparison of the method for detecting apoptosis of the present invention and the method relying on Annexin / PI co-staining based on the perspective of imaging effect

[0060]This example is carried out using A549 cells, but the apoptosis detection method of the present invention is not only applicable to A549 cells, only A549 cells are used as an example for illustration.

[0061] Cell culture: cells in the logarithmic growth phase were seeded in a 96-well plate with a certain number of cells per well (4×10 3 ~1×10 5 / well (according to different cell types) with a volume of 150 μL per well, cultured in a 37° C. incubator for 24 hours, and treated the cells after the confluence of the cells reached about 50% the next day.

[0062] Cell treatment: the cells were divided into 4 groups, 2 of which were control groups and 2 were apoptosis induction groups. The control group was not given any treatment, and the apoptosis induction group was induced by the apopto...

Embodiment 2

[0066] Example 2: The comparison between the apoptosis detection method of the present invention and the Annexin V / PI co-staining method based on the sensitivity of subgroup analysis

[0067] This example is carried out using Hela cells, but the apoptosis detection method of the present invention is not only applicable to Hela cells, only Hela cells are used as an example for illustration.

[0068] Cell culture: inoculate a certain number of cells per well in a 24-well plate (5×10 4 ~2×10 5 / well, according to different cell types), the volume of each well is 500 μL.

[0069] Cell treatment: The cells were divided into 6 groups, 2 of which were control groups, 2 were starvation treatment groups, and 2 were apoptosis induction groups. The control group was not given any treatment, the starvation treatment group was cultured with PBS instead of cell culture medium for 20 h, and the apoptosis induction group was induced with the apoptosis inducer staurosporine (STS), that is, c...

Embodiment 3

[0073] Example 3: Research on the Data Validity of the Cell Apoptosis Detection Method of the Present Invention

[0074] This example acts on Hela cells and A549 cells. Each type of cell is divided into 16 groups, of which 8 groups are the control group, and 8 groups are the starvation treatment group. The starvation treatment group uses HBSS instead of cell culture medium to incubate the cells for 20h, and then uses the staining method of the present invention in Example 2 for staining . The 16 groups of cell samples subjected to cell staining were detected and analyzed by flow cytometry to calculate the mean ± 3SD interval and Z'factor. Among them, the data validity test results of Hela cells and A549 cells are as follows: Figure 3-6 As shown, there were no outliers in the results. For Hela cells, mean±3SD (early apoptosis in control group) is [3.53,4.71], mean±3SD (early apoptosis in starvation treatment group) is [35.16,40.95], mean±3SD (late apoptosis in control group...

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Abstract

The invention relates to a cell apoptosis detection method and a cell apoptosis detection kit, comprising the following steps: staining a cell sample to be tested with a first fluorescent dye and a second fluorescent dye to obtain a stained cell sample, and then staining the stained cell sample The sample is subjected to fluorescence detection and analysis to obtain the apoptosis result of the cell sample to be tested; wherein, the first fluorescent dye is R-C 6 ‑D(OMe)E(OMe)VD(OMe)‑FMK, R is a fluorescent group; the second fluorescent dye can stain late apoptotic and necrotic cells; the emission wavelength of the first fluorescent dye and the second fluorescent dye different from each other. This detection method can quickly distinguish early apoptotic cells, late apoptotic cells, necrotic cells and normal living cells, and has a higher sensitivity for distinguishing cell subgroups, which can be used for high-throughput screening analysis, and the difficulty of image target segmentation lower.

Description

technical field [0001] The invention relates to the technical field of cell detection, in particular to a cell apoptosis detection method and a cell apoptosis detection kit. Background technique [0002] Apoptosis is a fundamental biological phenomenon of cells that plays an essential role in the removal of unwanted or abnormal cells in multicellular organisms. It plays an important role in the evolution of organisms, the stability of the internal environment, and the development of multiple systems. Apoptosis is not only a special type of cell death, but also has important biological significance and complex molecular biological mechanism. [0003] At present, there are many methods for detecting cell apoptosis, including Annexin V / PI composite analysis method, TUNEL method, DNA ladder method, mitochondrial membrane potential analysis method and many other methods. For example, glutathione sulfur transferase (GST) is coupled with human annexin V (Annexin V) to form a reco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6458G01N2021/6439
Inventor 张必良马林
Owner GUANGZHOU RIBOBIO
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