Analyzing DNA Damage and Repair in Cancer Cells

Immunofluorescence assay was performed for checking the BRCA1 and H2AX foci formation in cancer cells and in the cells which were treated with 0.035% MMS (methyl methane sulfonate) to check the repair activity using the following protocol: 5 × 10⁴ cells were seeded on collagen-coated coverslips in each well of a six-well plate. At 80% confluency, the cells were washed with 1X PBS and fixed in 4% paraformaldehyde, followed by permeabilization with 0.3% triton-X 100 in 1X PBS for 10 min each. Blocking was performed for 1 h with 5% FBS in 1X PBS at room temperature. The cells were incubated with primary antibodies overnight at 4°C in humid conditions, followed by the corresponding secondary antibody for 1 h at room temperature. After each antibody treatment, the cells were washed three times with 1X PBS. The coverslips containing cells were mounted on a glass slide with 10 ul of PD-DAPI, and imaging was carried out using an EVOS-M5000 microscope. For checking the repair activity, 0.035% MMS was added and incubated at 37°C for 15 min. After 15 min, the MMS was washed off with 1X PBS 2–3 times and released in fresh recommended culture media. The cells were fixed at 0 and 24 h after recovery. The cells without MMS were maintained as a control.