Automatic high-content imaging analysis system application - oncology research: automatic analysis of cell cycle and apoptosis

Automatic high content imaging analysis system application

—— Oncology research: automatic analysis of cell cycle and apoptosis

  Summary

  Monitoring the state of the cell cycle and apoptosis is a very important indicator in oncology research and treatment.   Compared with traditional detection methods, such as flow cytometry FACS , the fully automated high-content imaging analysis system can fully automate the imaging of cells in situ (without digestion and suspension) and perform multi-parameter analysis at the individual cell level. Image acquisition and analysis MetaXpress ^TM software having a series of common and simple application analysis module. AcuityXprss ^TM cell biology analysis software provides visual management and data mining of massive data generated by high-content imaging analysis.

  The cell cycle analysis module in MetaXpress software is an application module developed specifically for cell cycle and apoptosis analysis [Fig 1] . The module uses Adaptive Background Correcton ^{TM (} ABC ) to eliminate background non-uniformity caused by sample preparation and other factors, and to accurately distinguish cells from the background. This module allows for the analysis of cell cycle status in cells stained with a simple homogeneous DNA dye (eg DAPI/Hoechst/PI ) [Fig 2-3] and can also be analyzed with more accurate mitotic and / or apoptotic markers [ Fig 1] .

Fig 1:   Cell cycle analysis module interface

Fig 2. Determine the G0/G1, S, G2 phase by setting the threshold in the 2N and 4N bimodal maps according to the intracellular DNA content-total fluorescence intensity.

Fig 3. Under the labeling of monochromatic DNA dyes, the threshold of cells in the dividing phase can be set according to the degree of chromosome aggregation-average fluorescence intensity in each cell, and the cells in the dividing phase can be determined, and the 2N/4N parameters can be combined to further determine the division. Early Early M and late Late M.

  In the example below, we will analyze the effect of cell cycle and apoptosis on cells after treatment with positive compounds. We will use the cell cycle module in MetaXpress software to demonstrate a fast, simple, and flexible approach to cell cycle and apoptosis analysis.

Materials and methods

Sample Preparation

1. Prostate cancer Du145 cells were seeded at 8,000/ well in 96- well plates.
2. The cells were treated with paclitaxel Taxol and staurosporine at a concentration gradient for 24 hours.
3. The cells were fixed and used Hoechst33342 ( Invitrogen ), anti-phospho Histone H3ser10   ( Upstate )( Texas Red   Marked secondary antibody)   And anti-cleaved PARP   ( Cell Signaling )   ( FITC labeled secondary antibody   )dyeing.

Image acquisition

1.   Use ImageXpress Micro high connotations imager device, comes through MetaXpress Protocol standard software, for automatic 96-well plate were acquired with 10 flat-field image half apochromatic objective 20 and a half times the flat field apochromatic objective.

2.   Obtain 4 fields of view images per hole .

Analyze acquired images using the cell cycle module

1.   Image of selected marker DNA : Hoechst   Label the nucleus to reflect DNA content and structure.

2.   Set the size range of the nucleus and the contrast value with the background fluorescence intensity.

3.   Set the dividing cell parameters: defined by staining the defined DNA intensity or mitotic marker ( anti-phospho Histone H3ser10 ).

4.   Cells in an apoptotic state are set by an anti-cleaved PARP .

5.   Preview the analysis results and adjust the analysis parameters interactively.

6.   Automated analysis of all images.

data processing

1.   Analytical data was imported into Excel software for analysis.

2.   The data was mined using the high-content bioinformatics software AcuityXpress , including curve fitting, IC50/EC50 calculations, and Hit selection .

Fig 4. Image analysis using the cell cycle module to analyze the cycle state of the cells based on DNA content and structure. The comparison of the analysis result image with the original image: a) 20X original picture, b) 20X analysis picture, c) 10X original picture, d) 10X analysis picture.

Fig 5. In the case of 10x and 20x objective lens acquisition or different staining marker analysis, the drug efficacy results after drug treatment were consistent. DNA Avg. Intensity or DD means the use of DNA average fluorescence intensity to define dividing cells. H3s10 indicates that dividing cells are defined by a specific cleavage marker (anti-phospho Histone H3ser10). A indicates apoptosis Apoptosis, and M indicates split mitosis.

in conclusion

1.   Cell cycle analysis module for accurate nuclear and cell status analysis

2.   The cell cycle analysis module has simple parameter settings and operational aspects. The parameters can be adjusted through column and scatter plot interaction.

3.   The final data obtained with 10x and 20x objective lenses were consistent.

4.   DNA fluorescence intensity was used to define dividing cells and cleavage-specific markers to define dividing cells. The results of the two methods were consistent.

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