Radiation Biology

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SU_36_2363 - Co-Localization Analysis Method for High Content Screening (HCS) Measurement of Radiation Induced DNA Damage Response

Sunday, October 21
1:15 PM - 2:45 PM
Location: Innovation Hub, Exhibit Hall 3

Co-Localization Analysis Method for High Content Screening (HCS) Measurement of Radiation Induced DNA Damage Response
J. Feng1, Y. Sa1, Z. Huang2, and Y. Feng3; 1Department of Biomedical Engineering, Tianjin University, Tianjin, China, 2Saint Thomas Rutherford Hospital, Murfreesboro, TN, 3East Carolina University, Greenville, NC

Purpose/Objective(s): HCS is commonly used in studies of DNA damage response. Conventional quantification method is γH2AX foci counting, which needs manual adjustment and preset parameters, usually be regarded as imprecise, time-consuming, poor repeatability and its accuracy is often affected by DNA damage degree. This study was to develop a robust automatic alternative method for the quantification.

Materials/Methods: A549 cells were grown in 96-well plates, then treated with 5 μM of MS-275 or DMSO for at least 24 hours before exposure to 10 Gy in one delivery with 6 MV beam of Clinac 600 CD. After further culturing for 60 min, cells were fixed and then labeled with fluorescent antibodies. Images were acquired using IN Cell Analyzer 2200 with a 40× objective lens. Regions of nuclei were shown by DAPI staining in blue channel. Images of DNA double-strand break foci (γH2AX) were obtained from green channel by labeling with Alexa Fluor A488. Images of DDR (53BP1) foci were obtained from red channel by labeling with Alexa Fluor 546. An automatic quantification algorithm was developed for foci intensity measurement on the images. The summation of foci intensity in one nucleus, represented by Isum, is calculated with equation (1) Isum = ∑(i,j)S I(i,j) (1) where S is the region of integration, I(i,j) is the intensity of pixel (i,j) in S. γH2AX and 53BP1 are co-localized in one position when pixel intensities of this position are greater than zero in both green and red channels. We used a ratio, Ra, to represent the radiation-induced DNA damage repair capacity. Ra is given by equation (2) Ra = ∑(i,j)SG IR(i,j) / ∑(i,j)S IG(i,j) (2) IR is the intensity of this pixel in red channel, IG is the intensity of this pixel in green channel, and is the region of pixel intensity greater than zero in the green channel SG={S|IG(i,j)>0}. With this method, only the pixel intensity of 53BP1 foci co-localized with γH2AX was calculated. Comparative study of the new method with currently popular method of foci counting was conducted.

Results: Table 1 showed that foci numbers of γH2AX and 53BP1 between the cells from experimental and control groups were similar. But the pixel intensities of the foci were much different. Pixel intensity of γH2AX foci of the cell from experimental group was much higher than that from the control group, and trend of pixel intensity variation of 53BP1 foci was opposite, which in agreement with previous reports. MS-275 can increase DNA damage and inhibit the damage repair. It showed that foci counting method has possibility of yielding false negative when DNA damage was severe, and the integral of foci pixel intensities demonstrated better recognition. Table 1 Comparison of different quantitative methods on foci quantification in DNA damage
Foci number(d1) Integral of foci intensities(d2)
Nucleus A 46 522845
Nucleus B 43 920712
%diff [|d1-d2|/min(|d1|,|d2|)] 6.97 76.09

Conclusion: The new method provides a useful index for quantification of foci formation and DNA damage and detection accuracy is barely affected by the foci density.

Author Disclosure: J. Feng: None. Y. Sa: None. Z. Huang: Research Grant; NIH. Board Member; East Carolina University.

Jingwen Feng, MS, BS

Tianjin University

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