This method can help answer key questions in the DNA repair field such as how DNA repair complex formation, localization, and resolution are regulated. The main advantage of this technique is that it can unambiguously detect changes and repair complex localization and kinetics. In addition to these undergraduate researchers in my lab, three graduate students will perform the experiments.
That will be Changkun Hu, Dalton Dacus, and Stephen Walterhouse. To begin, grow U2OS/DR-GFP cells on a 10 centimeter tissue culture plate until they are 85 to 90%confluent. Then remove the medium, add three milliliters of EDTA, and incubate the cells at room temperature for three minutes.
Replace the EDTA with one milliliter of trypsin and incubate the cells at 37 degrees Celsius for five minutes. Then add five milliliters of DMEM to neutralize the trypsin. After counting the cells, seed six times 10 to the third cells per well in 200 microliters of medium in a glass bottom 96-well plate.
Alternatively, seed four times 10 to the six wells in eight milliliters of medium on 12 millimeter cover slips placed in a 10 centimeter plate, then grow the cells over night at 37 degrees Celsius and 5%carbon dioxide. To induce double strand breaks, or DSBs, replace the medium with hydrogen peroxide diluted to a concentration of 25 micromolar with DMEM plus 10%FBS and incubate the plate at 37 degrees Celsius for one hour. Wash the cells three times with 1x PBS, then add fresh DMEM.
To fix the cells after incubating them, use PBS to wash the plate three times. Then use a needle and forceps to carefully remove a cover slip from the 10 centimeter plate for each time point and place it in a single well of a 24-well plate. Add 4%PFA and incubate the cells for 15 minutes.
Then us PBS to wash the cover slips three times. To permeabilize the cells, add 0.5%non-ionic detergent and PBS. Incubate the cells at room temperature for 15 minutes.
Then wash the cells again with PBS three times. Next add 3%BSA and 0.1%non-ionic detergent diluted in PBS to the cover slips in a 24-well plate and incubate the plate at room temperature for one hour. Then add primary antibodies that target the repair protein of interest and incubate the samples at room temperature for one hour.
After washing the cells with PBS three times, add DAPI and incubate the plates at room temperature for five minutes. Then incubate the samples with secondary antibodies. After washing the cells as before, use mounting reagent to mount the cover slips cell side down onto slides.
Carefully press on the cover slips and use a wipe to soak up any excess mounting reagent. Seal the cover slips with fast drying transparent nail polish and ensure a complete seal of the cover slip with the slide. Take confocal microscope images by focusing on the DAPI channel.
To define the nuclei in immunofluorescence images, open the confocal images by dragging the image file into the ImageJ window or by selecting bio-formats importer from the plugin toolbar in ImageJ. Then select split channels. Under color options select colorized from the dropdown menu.
Select OK to open the DAPI and histone H2AX images as separate windows and choose the DAPI image. Then select the image toolbar in the adjust threshold option to select the nuclei. Next, adjust the image threshold by sliding the bottom bar of the threshold window completely to the right.
Adjust the top bar until the nuclei appear completely red with distinct outlines and the background is black. Then select the analyze toolbar and the analyze particles option. Input the minimum size of the nucleus.
From the show dropdown menu select outlines then select the add to manager option. Click OK to open an ROI manager window. From the ROI manager window assign numbers to the nuclei by selection.
To use ImageJ to quantify H2AX foci and immunofluorescence microscopy images select the H2AX foci window. Then select process toolbar and choose find maxima to find foci within the nuclei. From the output type dropdown menu choose the single points option and select the preview point selection to visualize the maxima foci.
Then input a noise tolerance value depending on the florescence intensity of the image. From the ROI manager window select the nuclei for which H2AX foci must be quantified. Select measure to measure the number of maxima foci within the selected nuclei.
Copy the raw intensity values and paste them into software for data analysis. After seating cells on 96-well plates with cover slips as demonstrated earlier in this video, induce double strand breaks by using a lipid-based transfectionary agent to transfect cells with the I-SceI expression vector. To acquire imagine after washing vix permeabilized and blocked cells as shown earlier, add antibodies against gamma H2AX and repair protein of interest.
With 1x PBS wash the 96-well plate cover slips three times. Then incubate the 96-well plate cover slips with secondary antibodies. Finally, after identifying the cells that have a large nuclear gamma H2AX focus, manually count those that also show colocalization with the repair protein of interest.
This figure shows a noise discrimination of 90 and marks the correct number of foci. Nuclei on the edge and foci outside the nuclei are not counted during the quantification. The panel shown here depicts a low noise tolerance of 50.
Numerous maxima are identified outside the nucleus and within the nucleus. A high noise tolerance of 220 is found in this cell. Only a single foci was detected.
Colocalization of a gamma H2AX focus and a repair protein of interest is depicted in this figure. An untransfected cell is shown here, and a cell with non-nuclear gamma H2AX focus is shown in the upper right. At higher magnification a clearer picture of a true interior focus and a non-nuclear or exterior focus can be distinguished.
Finally, an example of a nuclear gamma H2AX focus without colocalization of a repair protein is shown here. Once mastered, this technique can be done in a few days if it is performed properly. Following this procedure other methods like co-mutor precipitation can be performed in order to identify interactions among repair proteins of interest.
After watching this video, you should have a good understanding of how to observer the formation and resolution of DNA repair complexes by inducing transient double strand breaks in DNA. You will also learn how to unambiguously distinguish mislocalization of repair factors from delayed recruitment by inducing long-last lesions.
