20.109(F22):M1D4

Introduction

Protocols

Part 1: Analyze γH2AX images by counting foci

In addition to the above analysis, you will also use ImageJ to enumerate the γH2AX foci present in the nuclei of the treated cells. If you would like to review concepts used in this code, please review a protocol written by researchers at Duke University outlined here.

In this analysis, you will calculate the average foci per nuclei, indicating double strand breaks (γH2AX staining).

1. To begin, ensure that all of your H2AX images are in individual folders indicating the condition.
2. Download AnalyzeH2AX_batch script here.
3. Identify fluorescence intensity threshold values that will identify the nucleus in the DAPI channel.
   • Open a representative image in ImageJ.
   • Examine the first image in the stack, which corresponds to the DAPI channel.
   • Go to Image--> Adjust --> Threshold
     • Check the box for "Dark Background"
     • Make sure the cell nuclei are highlighted in red.
     • Adjust the threshold values to properly identify the majority of the cells' nuclei.
     • Record the threshold values, and repeat with other conditions to identify a representative threshold value.
4. Run the ImageJ macros to use fluorescence intensity to identify all the cells (DAPI channel) and cells with a significant amount of γH2AX sites (FITC channel).
   • In ImageJ, go to Plugins--> Macros--> Run, and click on the AnalyzeH2AX_CountMaximabatch script that you downloaded.
   • In the next window, choose the folder containing your .tif image stacks for the chosen condition, and click "Open."
   • You will be prompted to "Enter result file name" to name your spreadsheet for this analysis. Do so, and click "Ok".
   • In the dialog box titled "Choose Intensity Threshold Values," type in all the corresponding threshold values you have chosen, and click "OK."
   • Please wait for the script to run through all your images. In the end all the image files will pop up, along with the "drawings" that show where it identified cells in your images
   • On the "Drawing" windows that popped up, similar to the images shown here, each area identified by your fluorescence thresholds are assigned a number. Each of those numbers are listed in the corresponding Excel spreadsheets, followed by the information assessed by the ImageJ script.
   • For the DAPI channel, most of the identified regions correspond to a single nuclei. For the FITC channel, one may argue that for a cell having a significant amount of double strand breaks, the entire nucleus (rather than a few spots) shows up as being identified by the intensity thresholds provided.
   • The script will output resulting Excel files into your image folder. The Excel files contain raw data of each region of interest identified by the fluorescence intensity thresholds you chose.
   • You may use the Excel files to assess how foci are identified in each nuclei. To do this, divide the Raw Integrated Density (RawIntDen) for each nuclei by 255 (each maxima representing a foci has a value of 255) to identify the number of foci in each nucleus. You can now average the number for each image in the condition.
   • Repeat these steps for the additional conditions to quantify your results.

In your laboratory notebook, complete the following:

• How does the data obtained in the two analysis approaches compare? Are the results the same? Different?
• Which analysis approach best represents the raw data images? Why?

Part 2: Participate in group paper discussion

To further help you in preparing your Data summary, we will discuss how similar data are presented in a publication from the Engelward laboratory.

Weingeist, D. M., et al. "Single-cell microarray enables high-throughput evaulation of DNA double-strand breaks and DNA repair inhibitors." Cell Cycle. (2013) 126:907-915.

From the Introduction

Consider the key components of an introduction:

• What is the big picture?
• Is the importance of this research clear?
• Are you provided with the information you need to understand the research?
• Do the authors include a preview of the key results?

From the Results

Carefully examine the figures. First, read the captions and use the information to 'interpret' the data presented within the image. Second, read the text within the results section that describes the figure.

• Do you agree with the conclusion(s) reached by the authors?
• What controls were included and are they appropriate for the experiment performed?
• Are you convinced that the data are accurate and/or representative?

From the Discussion

Consider the following components of a discussion:

• Are the results summarized?
• Did the authors 'tie' the data together into a cohesive and well-interpreted story?
• Do the authors overreach when interpreting the data?
• Are the data linked back to the big picture from the introduction?

In your laboratory notebook, complete the following:

• Based on your reading and the group discussion of the article, answer the questions above.

Reagents list

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