20.109(F19):Complete CometChip experiment (Day5)

From Course Wiki
Revision as of 14:39, 23 September 2019 by Becky Meyer (Talk | contribs)

Jump to: navigation, search
20.109(F19): Laboratory Fundamentals of Biological Engineering

Fa19 20109 Banner image.png

Fall 2019 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Measuring genomic instability        2. Modulating metabolism        3. Testing chemical probes              


Introduction

As a brief reminder, two antibodies were used in the H2AX assay. The first antibody, or primary antibody, was anti-γH2AX and raised in a mouse. The secondary antibody was anti-mouse and raised in a goat, more importantly, this molecule is conjugated to a fluorescent dye tag called Alexa Fluor 594. The Alexa Fluor 594 tag is a bright, red fluorescent dye that is excited at 594 nm. To visualize the abundance of double-strand breaks in your H2AX assay samples, we will use fluorescence microscopy.

Diagram of a fluorescence microscope.

In fluorescence microscopy the specimen is illuminated with a wavelength of light specific to the excitation of the fluorescent tag used to target the feature of interest. The excitation wavelength is absorbed by the fluorescent tag, which causes it to emit light at a longer, less energetic wavelength. Typically, fluorescence microscopes used in biology are an epifluorescence type with a single light path (the objective) for excitation and emission detection, as depicted in the diagram above.

Fluorescence, or epifluorescence, microscopes are composed of a light source, an excitation filter, a dichroic mirror, and an emission filter. The filters and the dichroic mirror are specific to the spectral excitation and emission characteristics of the fluorescent tag. To visualize fluorescence, light at the excitation wavelength is focused on the sample. The light emission from the sample is focused by the objective to a detector.

Today you will see a demonstration concerning how to image the samples in your γH2AX assay and complete the data analysis.

Protocols

Part 1: Separate CometChip 'tails' using gel electrophoresis

  1. Remove your CometChip from the 1X PBS and use a kimwipe to dry the GelBond side.
  2. Carefully move your CometChip to the gel electrophoresis station in the 4 °C cold room.
  3. Place your CometChip on the raised center region of an electrophoresis box.
    • Double-sided tape was applied to the gel electrophoresis box. Be sure you lay your CometChip on the tape strips and lightly press down with a pipet tip to ensure it is secure.
  4. Add enough of the alkaline electrophoresis buffer to the gel electrophoresis box to cover your CometChip.
  5. Leave the CometChip in the alkaline electrophoresis buffer (aka unwinding buffer) for 45 minutes.
  6. To separate the damaged DNA into 'comets', electrophorese for 40 min (at 16 V, or 1 V/cm).
    • It is important that the electrophoresis occur at 300 mA. To maintain the appropriate current, the volume of electrophoresis buffer may need to be adjusted. The teaching faculty will assist you in adding/removing electrophoresis buffer such that this value is reached.
  7. Carefully remove your CometChip from the electrophoresis box and place it in a dish.
  8. Obtain an aliquot of neutralization buffer from the front laboratory bench.
  9. Wash your CometChip by adding enough neutralization buffer to cover (~10-12 mL) and incubate for 5 min at room temperature.
    • Repeat this step for a total of 3 washes.
  10. Add the SYBR gold DNA stain to your CometChip and carefully move it to the 4 °C cooler.

The teaching faculty will image your CometChip and provide the images to you in the next laboratory section.

Part 2: Analyze gamma-H2AX images

Please obtain your images from the instructors. There are two folders--let's begin with the one named "Two channel images" and save the other one for the next section. Three sets of images (i.e. image stacks) were taken per condition, and each image stack contains images from two channels--DAPI (blue) and Texas Red (red). Remember that the secondary antibody you used for the gamma-H2AX staining was conjugated to an Alexa594 fluorophore, which emits red light. For each image stack, you will use ImageJ to 1) identify the location of the nuclei using the DAPI channel and 2) quantify the total gamma-H2AX fluorescence in the Texas Red channel at locations specified by the DAPI channel.

Identify intensity thresholds for DAPI channel

Example of thresholding cell nuclei using ImageJ

The first thing you will need to do is identify intensity thresholds that will properly identify the cell nuclei in all the images. To be consistent and fair in analyzing fluorescence images, it is good practice to use the same intensity thresholds on all the images.

  1. Open ImageJ
  2. Open one image stack from the no treatment condition.
    • The first image you see is the DAPI channel
    • If you scroll to the right, the second image in the stack is the Texas Red (gamma-H2AX) channel.
  3. While the image is on the DAPI channel, go to Image -> Adjust -> Threshold.
    • A threshold window should pop up
    • 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.
    • Jot down the threshold values.
  4. Repeat this process for one image from each condition and cell line, and settle on threshold values for the DAPI channel that you will then use to analyze all the images. Write these values in your notebook.
    • It is best to define the lower threshold value based on your images, and set the upper threshold value as 256, which is the maximum possible intensity value for a 8-bit image.
    • You can type in threshold values by clicking on the "Set" button in the Threshold window.
  5. Close all open images (File -> Close All).

Test gamma-H2AX quantification on one representative image

  1. In ImageJ, open one image to test the FITC quantification protocol
  2. Split the image stack into two separate images
    • Go to Image -> Stacks -> Stack to Images
    • The DAPI image will have "-0001" as a suffix in its title
    • The Texas Red (gamma-H2AX) image will have "-0002" as a suffix in its title
  3. Duplicate the DAPI image and turn it into a mask to identify nuclei locations
    • Click on the DAPI image
    • Go to Image -> Duplicate, and click OK on the default title
    • Set the thresholds you chose on the duplicated DAPI image to identify nuclei
      • Go to Image -> Adjust -> Threshold
      • Check the box for "Dark Background"
      • Click on the "Set" button and type in your threshold values (use 256 for the upper threshold level).
    • Go to Process -> Binary -> Convert to Mask
      • This makes the image black and white, where the white areas should correspond to nuclei locations.
  4. Use the newly created mask to identify locations on the Texas Red channel in which to quantify the gamma-H2AX signal
    • Go to Analyze -> Set Measurements
      • In the Set Measurements window, make sure the following boxes are checked: Area, Mean gray value, Min & max gray value, Shape descriptors, Integrated density, Display label
      • In the "Redirect to" field, scroll and select the Texas Red image (suffix -0002). Then press OK.
        • This will direct ImageJ to the Texas Red image to analyze the metrics you selected in the areas identified by your mask. This will give you information about the gamma-H2AX signal in each nucleus.
  5. Run the analysis by selecting Analyze -> Analyze Particles
    Example of areas identified by intensity thresholds for DAPI channel in ImageJ
    • In the "Size" field, type 200-Infinity. This will eliminate small, extraneous particles that do not correspond to nuclei.
    • "Circularity" can remain at default values: 0-1
    • "Show" should say "Outlines"
    • Make sure to click the following options: Display results, Exclude on edges, Summarize
    • Press OK to complete.
  6. A window will pop up showing outlines of each nucleus the software identified based on the thresholds you defined. Each identified area is labeled with a red number, corresponding to the left column of the data shown in the "Results" window.
  7. Take a look at the "Results" window to see the results of the analysis. It is good practice to validate the numerical results by comparing them to what you see in the images.
    • The definition of the various measurements performed can be found on the ImageJ website here.
    • Does the nucleus with the largest "Area" correspond to the biggest nucleus you see in the drawing? The area here is in units of square pixels.
    • The RawIntDens field is the total intensity (sum of the intensity of all the pixels) of the corresponding region. Does a region with a high total intensity value correspond to a cell with a high gamma-H2AX signal? Click on the Texas Red image to double check.
  8. Close the "Results" window and do not save the data, as you will run the analysis on all the files together next.
  9. Close all open windows in ImageJ (File -> Close All)

Quantify gamma-H2AX signal in all images

  1. Ensure that all of your H2AX images are in one folder.
  2. Download AnalyzeH2AX_FITCintensityBatch_Fa19 script here. (Right click on the link and download the file into a folder where you can find it.)
  3. In ImageJ, go to Plugins--> Macros--> Run, and click on the AnalyzeH2AX_FITCintensityBatch_Fa18 script that you downloaded.
  4. When the script prompts you to "Choose input folder," choose the folder containing all your .tif image stacks (folder named "Two channel images"), and click "Open."
  5. In the dialog box titled "Choose Intensity Threshold Values," type in the corresponding DAPI threshold values you have chosen, and click "OK."
  6. You will be prompted to name the resulting Excel file next.
  7. 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
  8. The script will output one Excel file into your image folder.
  9. Before closing any images, validate the results in the Excel file with the images in ImageJ
    • Choose a few representative images to verify.
    • Check the "Drawing" images and DAPI images to see if the nuclei were called correctly by your threshold values. For example, if two or more nuclei were counted as one region, throw out the data point on the Excel sheet. If the nuclei were consistently identified incorrectly, consider choosing new threshold values and repeating the analysis.
  10. When you are done validating the data on the Excel sheet, post your data under the "Raw gamma-H2AX Data" column on the class data page (20.109(F19):Class_data) and close all the image files (File -> Close All).
  11. To share your analyzed data with the class, download this Excel template, fill it in, and also upload it to the "Analyzed gamma-H2AX Data" column on the class data page (20.109(F19):Class_data).

Reagents list

  • alkaline electrophoresis solution: 0.3 M NaOH, 1 mM Na2EDTA, pH 13.5 (from Sigma)
  • neutralization buffer: 0.4 M Tris, pH 7.5 (from Sigma)
  • SYBR gold DNA stain (from ThermoFisher Scientific)

Navigation links

Next day: Perform quantitative image analysis for the high-throughput genome damage assay

Previous day: Perform immunofluorescence imaging for repair foci and expose cell for high-throughput genome damage assay