An Immunofluorescence-Based Method to Quantify Replication Stress in Cancer Cells

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