This protocol allows for in cellulo quantification of DNA and resection following DNA damage. This technique's main advantage is the cell cycle marking which restricts the cells to S and G2 phase, ensuring the bromodeoxyuridine signal results from DNA and resection. Demonstrating the procedure will be Jean-Yves Masson, a principal investigator, and Sofiane Y.Mersaoui, a post-doctoral researcher.
Working under a sterile culture hood, place a single coverslip in each well of a six-well plate for as many conditions needed. Plate approximately 150, 000 HeLa cells for transfection or drug treatment. On day three, following the overnight incubation with BrdU at a final concentration of 10 micromolar, irradiate the plates with a total dose of five gray of x-ray irradiation.
After irradiation, return the plates for incubation at 37 degrees Celsius in a 5%carbon oxide humidified incubator for three hours. For pre-extraction, aspirate the medium and carefully wash the cells twice with PBS. After removing PBS, add two milliliters of pre-extraction buffer A and incubate the cells at four degrees Celsius on ice for 10 minutes.
After 10 minutes, aspirate buffer A, add two milliliters of cytoskeleton stripping buffer B, and repeat the incubation. Then carefully aspirate buffer B and wash the cells with PBS. After aspirating PBS, fix the cells by adding two milliliters of 4%of paraformaldehyde under the chemical hood.
Following removal of the paraformaldehyde and washes with PBS, cover the coverslips with 100%cold methanol for incubation at minus 20 degrees Celsius for five minutes and wash the coverslips twice with PBS. For permeabilization, incubate the cells with two milliliters of PBS containing 0.5%Tritan X-100 at room temperature. After 15 minutes, wash the coverslips three times with two milliliters of PBS.
For immunostaining, add two milliliters of blocking buffer to each well. After one hour of incubation, add 100 microliters of primary antibody solution to each coverslip. Use tweezers to cover the coverslips with square parafilm and carefully position the parafilm to not create bubbles.
Then cover the plate in aluminum foil and incubate the primary antibody overnight at four degrees Celsius in a humidified chamber. On the following day, remove the parafilm squares and wash the coverslips three times with two milliliters of PBS. After the wash, add 100 microliters of secondary antibody solution on each coverslip and cover the coverslips with a square of parafilm as demonstrated.
Incubate the secondary antibody at room temperature for one hour and protect the plate from light using foil. Then wash the coverslips three times with two milliliters of 1X PBS. For nuclear staining, add two milliliters of DAPI solution to each coverslip and wash the coverslips with PBS.
Add 10 to 20 microliters of IF-specific mounting medium on microscope slides. Use the needle or fine tweezers to lift the coverslip from the bottom of the well. Carefully blot off excess liquid by tapping one edge gently on a paper towel and mount the coverslips on microscope slides.
For image acquisition and analysis, import these files into CellProfiler and analyze them using the speckle counting pipeline. Identify the primary object to identify the foci using the settings described in the text manuscript and measure the intensity. Representative images of 5-bromo-2-prime-deoxyuridine foci formation and proliferating cell nuclear antigen staining following irradiation are shown here.
The generated single-stranded DNA tracks were visualized as distinct foci. The identified foci were then quantified and expressed as the total integrated intensity of the bromodeoxyuridine staining in the nuclei. Co-staining showed differentiation between the short range resection due to non-homologous end joining and the long range resection of homologous recombination using an anti-PCNA antibody to identify cells going through the S phase.
PCNA is prominent in the nucleus and reaches maximal expression during the S phase of the cell cycle with quite an intense staining. The result in values are then plotted as a scatterplot to discriminate between PCNA positive and PCNA negative nuclei. The PCNA negative results are removed from the dataset to allow for BrdU intensity-based analysis because of the low BrdU signal regardless of the condition.
The PCNA negative nuclei harbor a basal integrated intensity of BrdU foci of 900 arbitrary units and this value reaches 1, 800 AU in PCNA positive nuclei. In the siRNA control condition, the integrated intensity of BrdU foci per nucleus is approximately 1, 500 AU in the PCNA positive nuclei. After an efficient knockdown of PARP-1 using an siRNA, a significant increase in the intensity of the BrdU foci to approximately 1, 900 AU was observed.
This technique can provide key insights into the first stage of homologous recombination, thus helping to identify if a protein is involved in the first step of the repair pathway. The incubation time for the pre-extraction buffers must be strictly kept. Over-exposure to these buffers will result in excessive cell detachment and increased background signal.
