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DNA Damage and Repair - Quantification of Sub-Cellular Events Using Automated Confocal Imaging

ment both over time and with increasing concentrations of H2O2, a known inducer of DNA damage.

Methods

Cell Culture

HT1080 cells (ATCC, CCL-121, human fibrosarcoma derived cell line) were grown in 37C, 5%CO2 humidified incubators according to the suppliers protocol. Cells were seeded at 10,000 cell/well into 96 well plates (Cat. No. 353219) and incubated overnight. H2O2 (Sigma, H-1009) was used to initiate DNA double strand breaks. Immediately prior to the experiment, H2O2 was diluted in pre-warmed media and dose-response curves were obtained by serial dilution. Cells were exposed to chemical insult for 10 minutes at room temperature after which they were washed and replenished with fresh media. To allow for DNA repair processes to occur, the cells were returned to the incubator and left for 30, 90, or 180 minutes. In some experiments, cells were left to recover for 16 hours. At those time points, cells were fixed for 10 minutes with pre-warmed formaldehyde, 3.7% in phosphatebuffered saline (PBS), washed with PBS and permeabilized with ice-cold 90% methanol (10 minutes). Cells were washed twice with PBS and blocked using 1% BSA (in PBS) for 30 minutes. For the detection of phosphorylated H2AX, the mouse monoclonal anti-phospho serine 139 H2AX antibody (Chemicon, 05-636) at 2 μg/mL in 1% BSA block was used. 53BP1 was detected using the anti-53BP1 rabbit polyclonal antibody (Novus Biologicals, NB 100-304) diluted as described for H2AX.

After 1h at room temperature, the primary antibody was removed and cells were incubated with Tween 20 (0.05% in PBS, 10 minutes). Two PBS washes prepared the cells for the addition of the secondary antibodies, 5 μg/mL Alexa Fluor 555-conjugated anti-mouse IgG (Cat. No. A21424) for H2AX and 5 μg/mL Alexa Fluor 488-conjugated anti-rabbit IgG (Cat. No. A1
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