Rabbit anti phospho histone h2a x ser139 antibody

40,000 4T1 cells were plated in a gelatin-coated 8-well chamber slide and allowed to settle for 24 hours. At 24 hours, media was replaced with media containing drug or MPM treatment. Cells were incubated with various drug or MPM treatments (Figure 3) for 72 hours and then irradiated with 5 Gy (CellRad). Three hours after irradiation, cells were washed 3× with PBS and formalin fixed. Formalin was removed by washing with PBS and then blocked and permeabilized using 5% Goat Serum and 0.3% Triton X-100. Following blocking and permeabilization, cells were incubated with a rabbit anti-Phospho-Histone H2AX (Ser139) Antibody (#2577, Cell Signaling Technologies) at a 1:800 dilution for 1 hour at room temperature in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in PBS). Next, cells were washed with PBS and incubated with a goat anti-rabbit IgG H&L (Cy 5) antibody (6564, Abcam) at a 1:1600 dilution in antibody dilution buffer in the dark for 1 hour at room temperature. Lastly, cells were once more washed with PBS and imaged using an EVOS FL Auto microscope at 40 × magnification. Using 40× EVOS images (> 150 cells/ treatment group in various images), DNA damage quantification was automated by cell profiler overlaying the DAPI stained nucleus with the Cy 5 stained γH2AX foci for a foci/cell count. To be counted, a focus had to be greater than 7 pixels.