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Introduction

Jayne Hesley | Applications Scientist | Molecular Devices

Assessing damage to DNA or chromosomes is frequently addressed with research applications because of its implications in diverse diseases including genetic mutations, cancer, and aging. DNA damage may occur spontaneously or result from exposure to ionizing radiation, environmental influences and contaminants, or chemical compounds. Previous research demonstrated that phosphorylation of histone H2AX on serine139 is an early and sensitive indicator of DNA double-strand breaks¹.

This study details our development of an automated cellular imaging assay for characterizing DNA damage effects of selected small molecules based upon immunofluorescent detection of phosphorylated histone H2AX. Specifically, our methods enabled quantitation of nuclear DNA damage in CHO, HeLa, or PC12 cells (not all data presented) in response to treatment with the chemotherapeutic agents Mitomycin C (MMC) and etoposide.

Cellular immunofluorescence assay for phospho-H2AX quantifies DNA damage

Cellular DNA damage can be visualized and quantified using commercially available reagents with the ImageXpress® Nano Automated Imaging System. A cellular immunofluorescence assay was designed using primary antibodies against phosphorylated H2AX (EMD Millipore) paired with an AlexaFluor labeled antispecies antibody (Life Technologies). Nuclei were counterstained with Hoechst 33342 and images were acquired on the ImageXpress Nano system using a 10X objective and standard DAPI and FITC filter sets (Figure 1).

Figure 1. DNA damage IF assay.

• Seed cells at 5000–7500 cells/ well in 384-well clear bottom polystyrene microplate
• Culture cells overnight at 37°C/5% CO ₂
• Treat cells with a 1:2 serial dilution of DNA-damaging compounds for 18-24 hours
• Fix with 4% formaldehyde for 20 minutes at room temperature
• Wash cells with PBS, then block and permeabilize cells with 1% BSA+ 0.1% Triton X-100 for 30 minutes at room temperature
• Add anti-H2AX primary antibody and incubate overnight at 4°C
• Wash three times with 1x PBS
• Add fluorescently labeled secondary antibodies + 16 µM Hoechst and incubate 1-2 hours at room temperature
• Wash cells three times with 1x PBS
• Acquire images on ImageXpress Nano system with 10X Plan Fluor objective and perform on-the-fly Cell Scoring analysis

On-the-fly cell scoring analysis efficiently identifies nuclei with damaged DNA

The CellReporterXpress^™ Automated Image Acquisition and Analysis Software includes a preconfigured Cell Scoring application module that can be easily configured to classify nuclei as positive or negative for a DNA damage marker as well as calculating the percentage of cells exhibiting DNA damage. The acquisition and analysis routines are coupled, enabling rapid review of both images and numerical data upon completion of the plate read. Additionally, due to the large field of view captured, imaging a single site with a 10X objective is sufficient to calculate accurate concentration response curves (Figure 2). Specifically, imaging a single field-of-view at this magnification captured >500 cells in the highest tested concentration and >2800 cells in wells treated with sub-toxic concentrations of Mitomycin C (MMC).

Figure 2. (A) Nuclei were imaged at 10X magnification and scored as positive or negative for the double-stranded break marker phospho-H2AX. This split view shows the fluorescently stained nuclei (red) and nuclei positive for the DNA damage marker (yellow/green) on the left and the cell scoring mask with all nuclei marked in grey and positive nuclei marked in yellow on the right. (B) The DNA damage response in CHO cells after 24 hour treatment with chemotherapeutic compounds. A dose response of the compound was calculated by plotting the percentage of phospho-H2AX positive cells versus MMC and etoposide concentrations.

Conclusion

The phosphorylation of histone H2AX is a sensitive indicator of double-stranded DNA breaks in mammalian cells. Here, we demonstrate the utility of the ImageXpress Nano system with CellReporterXpress software to easily automate quantification of DNA damage in CHO cells via an immunofluorescence cell based assay in multi-well microplates. The integrated image acquisition, on-the-fly analysis, and instant data visualization capabilities (Figure 3) of this cellular imaging system enables objective evaluation of concentration response effects on DNA damage, delivering improved data quality and throughput versus manual microscopy methods.

Figure 3. (A) DNA damage responses to MMC or etoposide treatment can be quickly assessed by reviewing integrated heat map visualizations of percent H2AX positive nuclei across the microplate. In this figure, the highest treatment of compound is in the top row, in 2 sets of quadruplicates. Wells containing a high percentage of H2AX positive nuclei show as red, and low % positive show green. (B) Data can also be visualized on a scatter plot with concentration of etoposide on the X axis and % positive cells on the Y axis.

Reference

1. Paull T.T., Rogakou E.P., Yamazaki V., Kirchgessner C.U., Gellert M., Bonner W.M..2000. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol. 10, 886-95.

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