Our protocol presents a highly efficient and easily reproducible method to quantify single stranded DNA, which is a marker of replication stress in a variety of cell lines. Additionally, its simplicity allows for high throughput applications and screens. In particular, this method allows for rapid quantification of a replication stress, a hallmark of several ovarian cancers.
It is also amenable to a host automatic analysis software, further increasing efficiency. Single stranded DNA is now being actively considered as a biomarker for response to chemotherapy targeting DNA repair pathways. Hence, this method is highly applicable in that scenario.
Replication stress exists beyond ovarian cancer contexts or BRCA1, BRCA2 deficient cancers. And thus, this method can be applied to a wide array of other cell types or disease contexts. To begin, make polylysine-coated cover slips by adding autoclaved 12 millimeter diameter cover slips and polylysine solution to a 50 milliliter conical tube, and place on a rocker for 15 minutes.
Aspirate the solution on a tissue culture hood. Wash the cover slips by adding sterile water, and place the tube containing the cover slips back on the rocker for five minutes. After washing the cover slips three times, aspirate water from the tube and spread the cover slips on a sterile dish.
Aspirate the remaining water and let them dry in the tissue culture hood for one hour or until no water droplets remain. Once dry, seal the dish with Parafilm and place it at 4 degrees Celsius. To prevent the detachment of the cells from the cover slips during the pre-extraction step, place one polylysine-coated cover slip in each well of a 24-well plate.
Once the OVCAR3 cells reach 70 to 80%confluency, aspirate the medium from the plate and wash the cells with 5 to 7 milliliters of PBS. Aspirate the PBS from the plate. After aspirating the PBS, add 1 milliliter of 0.25%trypsin and place the dish in the incubator at 37 degrees Celsius for 8 to 10 minutes or until cells lift off from the bottom of the plate.
Collect the cells with 5 to 10 milliliters of medium and add the cell suspension to a conical tube. Count the cells manually with a hemocytometer using standard procedures. Next, dilute the cell suspension, and obtain a number that will yield 70 to 80%confluency after three populations.
Add 1 milliliter of cell suspension on polylysine cover slips placed in the wells of a 24-well plate, and grow the cells in the culture medium under standard conditions. After one population doubling, aspirate the existing media from the plate and pulse the cells with 10 micromolar IdU for the two subsequent population doublings. To harvest the cells, replace the medium with ice cold 0.5%PBSTx on ice for five minutes.
For fixation of cells, aspirate PBSTx and incubate the cells for 15 minutes with 3%paraformaldehyde at room temperature. After three to four washes with PBS, keep fixed cells at 4 degrees Celsius until further use. After fixation, permeabilize the cells using 0.5%PBSTx on ice for five minutes, ensuring to cover the entire cover slip.
After washing the cells three to four times with 1 milliliter of 0.2%PBST at room temperature, aspirate the PBST and block the samples using 5%BSA made in PBS for 30 minutes at room temperature. For immunostaining, prepare a unified chamber by putting a wet paper towel on a flat bottom Tupperware. Cover the 24-well plate lid with Parafilm.
Place it in the humidified chamber and lay down the cover slips on the plate lid. Add 60 microliters of diluted anti-BrdU primary mouse antibody on the top of the cover slip. Alternatively, to reduce the antibody from drying and use less volume, place a drop of diluted antibody on the Parafilm and flip the cover slip onto it.
Then incubate for one hour at 37 degrees Celsius. After incubation, aspirate the primary antibody. Return the cover slips back to a 24-well plate and wash them with 0.2%PBST four times.
Add diluted secondary antibody on the cover slip as demonstrated earlier, and incubate for one hour in the dark at room temperature. Label a microscope slide and mount the cover slip on the slides with DAPI mounting medium. Store slides in the dark at room temperature for 24 hours if the mounting medium needs to be cured or hardened.
The nuclei derived from the untreated and treated cells with 0.5 millimolar hydroxyurea were stained and identifiable in the DAPI and IdU channels. The analysis of these images consists of quantifying the number of foci in each nucleus. The number of foci is proportional to the degree of replication stress.
It is important to consider the doubling time of the cell line of choice as the time of IdU pulsing may vary for longer or shorter doubling periods. Alternatively, we can probe cells for replication protein A to confirm the results and assess various replication stress responses in cells. This technique is used for many cell lines in tandem with any DNA damage-inducing agent to examine the accumulation of single-strained DNA.
Thus, this method is widely accessible and applicable.
