Difference between revisions of "20.109(F18):Analyze genomic instability experiment data and treat cells for sub-nuclear foci assay (Day5)"

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(Introduction)
(Part 2: Treat cells with H2O2 for sub-nuclear foci assay)
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#Prepare your H<sub>2</sub>O<sub>2</sub> dilution according to the calculations you completed in Step #1.
 
#Prepare your H<sub>2</sub>O<sub>2</sub> dilution according to the calculations you completed in Step #1.
 
#Retrieve the two 12-well plates that you seeded the previous lab from the 37 &deg;C incubator.
 
#Retrieve the two 12-well plates that you seeded the previous lab from the 37 &deg;C incubator.
 +
#Aspirate off the media in the wells.
 
#Add 1 mL of fresh media or media + H<sub>2</sub>O<sub>2</sub> to the appropriate wells according to the plate map.  
 
#Add 1 mL of fresh media or media + H<sub>2</sub>O<sub>2</sub> to the appropriate wells according to the plate map.  
 
#*Only the experimental wells will be treated with media + H<sub>2</sub>O<sub>2</sub>!
 
#*Only the experimental wells will be treated with media + H<sub>2</sub>O<sub>2</sub>!

Revision as of 19:55, 26 September 2018

20.109(F18): Laboratory Fundamentals of Biological Engineering

Fa18 20109 banner image.png

Fall 2018 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Measuring genomic instability        2. Modulating metabolism        3. Engineering biomaterials              


Introduction

In this module, you are assessing the role of DNAPKcs in the response to oxidative stress by evaluating DNA damage. The first experimental approach used the CometChip to measure DNA damage to increasing doses of H2O2. Though some degree of repair occurred during the treatment incubation, the full capacity of the cell to repair the damaged DNA was not measured given that the cells were lysed immediately after H2O2 exposure.

It is known from the literature that H2O2 treatment results in single-strand breaks in DNA (remember: the repair process is actually what generates the strand breaks in response to the damaged guanine!). When several single-strand breaks are present, it is possible that two single-strand breaks within close proximity to one another will result in a double-strand break. To assess this you used the γH2AX assay to visualize and quantify double-strand breaks. In addition to evaluating DNA damage in response to H2O2 treatment, you will use the γH2AX assay to consider recovery capacity (or the ability to repair damaged DNA) in the +DNAPKcs cells compared to the -DNAPKcs cells. To do this, the cells will be incubated in fresh media following H2O2 exposure.

Protocols

Part 1: Process microscopy images for biochemical testing experiment

First, download your team's CometChip images from the Class data tab or obtain images from an instructor. Create (or copy and paste) two folders of your team images--one for the enzyme treated CometChip and one for the buffer control CometChip, into the Documents\MATLAB\CometChip Analysis directory of the lab computer.

You will complete the following processes twice (stack images, optimize analysis parameters, measure tail lengths, export to Excel)--once for each CometChip. You should end up with one Excel file for each chip.

Stack images using ImageJ

  1. The script you will run will combine images from the same well into a stacked image and rename the files for the subsequent Matlab script to recognize them.
  2. Open ImageJ from Applications.
  3. Go to Plugins → Macros → Run...
  4. Select script "GenImageStacks_singleimage.txt" within the Documents\MATLAB\CometChip Analysis directory
  5. Choose the appropriate source directory that contains your image files.
    • Find in Documents\MATLAB\CometChip Analysis directory
  6. Create a destination directory by selecting New Folder and naming the folder appropriately (eg. "180927M059_H2O2_stacked").
  7. Click Choose.
    • ImageJ will create the stacked images in ~2 min.
    • Please do not hit any additional keys until this process is completed.
  8. Confirm that the stacks are in the destination directory.
    • Open the directory you just created, containing your stacked images.
    • You should see one .tif image stack per well.
  9. Close ImageJ.

Optimize analysis parameters using MATLAB

  1. Open MATLAB from Applications.
    • Be sure that "CometChip Analysis" is the current folder (double click on the "CometChip Analysis" folder from within the "Current Folder" window).
  2. In the command window, type in "guicometanalyzer" press enter to run the script.
  3. In the MultiWell Comet Analyzer window:
    • Check that the Pixel Calibration value is 1.61.
    • Check that the Image Rotation value is 0 if your tails are to the right. Image Rotation should be 180 if your tails are going to the left.
    • Check that the Array Spacing (μm) is 240.
    • Check that the Head Diameter value is 20.
    • Confirm the settings with the image below.
      Multiwell Comet Analyzer window.
  4. Click Browse.
    • Select the stacked image directory you created in the previous section.
    • Click Open.
      CometChip analysis showing differentiation between head and tail.
  5. From the image stack files loaded to the MultiWell Comet Analyzer window, choose ONE image that is expected to show DNA damage. You are just using this image to optimize the analysis parameters.
    • The chosen file should be highlighted.
    • Click Select.
  6. Click Analyze.
  7. Click "Yes" when the dialog box appears and asks if you want to run the program in "debug mode."
    • Please do not hit any additional keys until this process is completed.
    • In the MATLAB command window you will see the number of comets to be analyzed. The same number of figures should be generated.
  8. Review the generated images.
    • Ensure that the head of the comet is bracketed by a white line on the left, and a red line on the right. The tail should be bracketed by the red line and a blue line on the right. See the image to the right for an example.
    • You may need to adjust the "Tail Threshold" value so that the tail is identified appropriately and/or the "Head Threshold" value so that the head is identified appropriately.
    • Note: it is okay if some of the images do not appear correct according to the above criteria; however, the majority of your images should be correct.
  9. In the Command Window type "close all."
  10. If you need to adjust your parameters, repeat this process again until you find the optimal parameters for your analysis.
    • Find the guicometanalzyer window.
    • Adjust parameters as needed and repeat steps above by continuing to run in "debug mode."
    • Type "close all" when done, but leave Matlab open.

Measure head and tail lengths using MATLAB

  1. Find the guicometanalyzer window
    • If it has disappeared, run the script again by typing "guicometanalyzer" in the command window.
  2. In the MultiWell Comet Analyzer window, confirm the parameters as above.
    • Click Browse.
    • Select the stacked image directory you created with ImageJ.
    • Click Open.
  3. Highlight all stack files in the MultiWell Comet Analyzer window.
    • Click Select.
  4. Click Analyze.
  5. Click "No" when the dialog box appears and asks if you want to run the program in "debug mode."
  6. Confirm that there is a .txt file for each stack in your folder.
    • Be sure to keep MATLAB open.

Compile results and export to Excel

  1. In the command window, type "comettoexcel" and press enter to run the script.
  2. When prompted, select the directory containing all the txt files that were output by guicometanalyzer and click "Open."
  3. The script will compile all your data, calculate the median values for each well, and export the data in an Excel file.
  4. Next, a dialog box will prompt you to save the Excel file.
  5. Please choose an appropriate directory and file name to save your data.
  6. Click "Save" and ensure your data has saved correctly.

Post your final Excel spreadsheets on the Class data tab.

Part 2: Treat cells with H2O2 for sub-nuclear foci assay

Before you enter the tissue culture room, let your instructor know the recovery time that you will use in your experiment and confirm your calculations.

Fa18 M1D5 H2AX treatment plate map.png
  1. Calculate the dilution of H2O2 needed to have a final concentration of 1mM for each experimental well.
    • H2O2 stock concentration is 10 M. The teaching faculty prepared a 100 μM solution that will be used to prepare your treatment concentrations.
    • You will need 1 mL of media containing H2O2 per well.
  2. Upon entering the tissue culture room, ready your tissue culture hood with the supplies you will need to complete the experimental procedures.
  3. Prepare your H2O2 dilution according to the calculations you completed in Step #1.
  4. Retrieve the two 12-well plates that you seeded the previous lab from the 37 °C incubator.
  5. Aspirate off the media in the wells.
  6. Add 1 mL of fresh media or media + H2O2 to the appropriate wells according to the plate map.
    • Only the experimental wells will be treated with media + H2O2!
  7. Incubate in the 4 °C cooler for 20 min.
  8. Retrieve your 12-well plates from the cooler and aspirate the media from both plates.
  9. Immediately add 400 μL of 4% paraformaldehyde to Plate #1 to fix the cells.
  10. Incubate at room temperature for 10 min.
  11. During this incubation, add 1 mL of warm media to the remaining 12-well plate and move it to the 37 °C incubator for recovery.
  12. Collect the 4% paraformaldehyde in the correct waste stream using a P1000 pipet from Plate #1.
  13. Wash with 500 μL of 1X PBS.
    • Add 1X PBS then remove using a P1000 pipet. Collect the PBS in the correct waste stream.
    • Complete a total of 2 times. Leaving 1 mL of 1X PBS on the cells in the final wash.
  14. After your chosen recovery time has elapsed retrieve Plate #2 from the incubator and fix according to Steps #7 - #9, then wash according to Steps #11 - 12.
  15. Leave all wells with 1 mL PBS, parafilm the sides and move the fixed plates into the 4 °C cooler.

Reagents

H2AX assay

  • 1:1 Dulbecco's Modified Eagle's Medium (DMEM) : Ham's F12 medium (Gibco)
    • with 10% fetal bovine serum (Atlanta Biologicals)
    • 0.05 mM Non-essential amino acids (Gibco)
    • 100X antibiotic solution (Gibco)
      • 10,000 U/mL Penicillin
      • 10,000 U/mL Streptomycin
  • H2O2: hydrogen peroxide (Sigma Aldrich)
  • paraformaldehyde 4% (VWR)

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