This article details a method for quantifying replication stress in cancer cells using IdU incorporation and immunofluorescence techniques. The process involves fixing and permeabilizing cells, followed by antibody staining and fluorescence microscopy to visualize and count DNA foci.
Begin with cancer cells carrying IdU, a synthetic substitute of thymidine nucleotide, already integrated into their DNA. When these cells encounter replication stress, their DNA synthesis halts, revealing IdU within exposed single-stranded DNA regions.
Fix the cells and add detergent molecules to make the cell membrane permeable.
Add BSA to block non-target antibody binding sites.
Introduce anti-IdU antibodies that interact with IdU-labeled single-stranded DNA.
Add green fluorophore-labeled secondary antibodies that target anti-IdU antibodies. Remove the unbound antibodies.
Place the coverslip on a slide containing a mounting medium with a fluorescent dye — that stains the nuclei.
Under a fluorescence microscope, observe the blue nuclei.
The green fluorescence foci represent antibodies binding to the single-stranded DNA.
Count the number of foci to quantify the level of replication stress.
Add 1 milliliter of cell suspension on poly-L-lysine coverslips 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 5 minutes. For fixation of cells, aspirate PBSTx, and incubate the cells for 15 minutes with 3% paraformaldehyde at room temperature. After 3 to 4 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 5 minutes, ensuring to cover the entire coverslip. After washing the cells 3 to 4 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 humidified 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 coverslips on the plate lid. Add 60 microliters of diluted anti-BrdU primary mouse antibody, on the top of the coverslip.
Alternatively, to reduce the antibody from drying and use less volume, place a drop of diluted antibody on the parafilm and flip the coverslip onto it. Then, incubate for 1 hour at 37 degrees Celsius. After incubation, aspirate the primary antibody.
Return the coverslips back to a 24-well plate and wash them with 0.2% PBST 4 times. Add diluted secondary antibody on the coverslip, as demonstrated earlier, and incubate for one hour in the dark at room temperature. Label a microscope slide, and mount the coverslip 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.
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